Mesoporous permeation layers for use on active electronic matrix devices

ABSTRACT

The present invention provides improved synthetic polymer hydrogel permeation layers for use on active electronic matrix devices for biological assays. The permeation layers have a defined porous character, with mesopores in a size range between about 100 nanometers and about 1000 nanometers, and may also have micropores in the micrometer size range. The mesoporous synthetic hydrogel permeation layers demonstrate improved signal intensity and linearity characteristics as compared to nanoporous synthetic hydrogel permeation layers on active electronic matrix devices. In addition, the present invention also provides synthetic polymer hydrogel permeation layers which contain copolymerized attachment sites for nucleic acid probes or other biomolecules.

FIELD OF INVENTION

The present invention provides improved synthetic polymer hydrogelpermeation layers for use on active electronic matrix devices forbiological assays. The permeation layers have a defined porouscharacter, with mesopores in a size range between about 100 nanometersand about 1000 nanometers, and may also have micropores in themicrometer size range. The mesoporous synthetic hydrogel permeationlayers demonstrate improved signal intensity and linearitycharacteristics as compared to nanoporous synthetic hydrogel permeationlayers on active electronic matrix devices. In addition, the presentinvention also provides synthetic polymer hydrogel permeation layerswhich contain copolymerized attachment sites for nucleic acid probes orother biomolecules.

BACKGROUND

The following description provides a summary of information relevant tothe present invention. It is not an admission that any of theinformation provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to the invention.

By placing a plurality of nucleic acid probes on a surface, and exposingthe surface to a sample containing target nucleic acids, manyhybridization reactions may be carried out on a sample at the same time,simultaneously generating hybridization data for several target nucleicacids (the reverse dot-blot technique). Similarly, by immobilizingnucleic acids from several samples onto the surface, several samples maybe probed with the same oligonucleotide probe at the same time (thedot-blot technique). Originally, dot-blot and reverse dot-blothybridizations were carried out using nucleic acid probes crudelyblotted onto a nucleic acid-binding membrane or filter. In the past twodecades, several tools have been designed to place nucleic acid probesat defined locations in high densities on various types of surfaces(glass, polymers, silicon nitride, etc.) by methods such as physicaldeposition (e.g., ink-jet, microspray, pin deposition, microchanneldeposition) or by in-situ polymerization techniques (e.g.,photo-deprotection methods.) Such “microchip” based DNA arrays have beenof great interest in recent years due to their enormous ability tofacilitate rapid analysis of genetic information. Although very advancedtechniques are utilized to generate these types of arrays, they stillemploy parallel hybridization of DNA to the immobilized capture probesin a passive mode. In other words, the nucleic acids present in theentire sample volume interact with the entire array surface at the sametime, to the same extent.

In contrast, active electronic matrix arrays use an electric field tofacilitate the rapid transport and hybridization of DNA on microchips.In general, active matrix array devices contain an array ofelectronically addressable microelectrodes on a substrate, which provideelectric field control over a variety of biomolecular reactionsincluding DNA transport, hybridization and denaturation. By using theelectrodes to apply an electric field to a solution containing chargedmolecules, such as nucleic acids, the charge molecules can be rapidlytransported to and concentrated at the electrodes which are biasedopposite the charge of the molecules. This allows the transport ofnucleic acid probes or amplicons to the microlocations in a veryefficient and specific manner for binding to attachment moieties at themicrolocations (a process sometimes referred to as “programming” thelocations), allowing the generation of arrays for dot-blot or reversedot-blot formats. After the probes or amplicons are immobilized at themicrolocations, the electric field can again be used to rapidly directthe second hybridization assay component to the microlocation. Thus,electric field regulated hybridization is one to three orders ofmagnitude faster than passive hybridization under the same conditions,overcoming several of the limitations of passive hybridization.

These arrays, also known as active programmable electronic matrixdevices, or APEX devices, have been extensively described, e.g. in U.S.Pat. Nos. 6,051,380 and 6,245,508, incorporated herein by reference intheir entirety. In general, the devices comprise an array ofindividually controllable microelectrodes on a substrate, and optionallycomprise additional counter electrodes for opposite biasing. Themicroelectrodes are overlaid by a thin permeation layer, defining themicrolocations of the device above the microelectrodes. In addition tofacilitating the attachment of biomolecules by providing a matrix toaffix attachment moieties (e.g., streptavidin,) the permeation layerseparates the biomolecules from the electrode surface where hydrolysisand other potentially detrimental electrochemical reactions can occur.Although the permeation layer retards or prohibits the movement of thebiomolecules towards the microelectrode, the permeation layer issufficiently permeable to small molecules to permit ion exchange betweenthe electrode surface and the buffer medium, allowing an electriccurrent to flow. The active electronic matrix chips usually use electriccurrent and voltage conditions wherein electric current densities are atleast 0.04 nA/μm² (about 200 nA for an 80 μm diameter microlocation)and/or potentials sufficient to hydrolyze water. The electric currentdensity is defined as the electric current divided by the area of theelectrode used to support it.

Additionally, the effectiveness of the translocation of chargedbiomolecules such as nucleotide oligomers within anelectronically-driven system such as an active electronic matrix chipdepends on the generation of the proper gradient of positively andnegatively charged electrochemical species by the anode and cathode,respectively. For example, effective nucleic acid (i.e. either DNA orRNA) transport may be accomplished by generation of protons and hydroxylanions when the potential at the anode is greater than +1.29 V withrespect to a ‘saturated calomel electrode’ (SCE). The transportefficiency of charged molecules increases with increasing currentdensity, thus driving the desire for operation at higher voltage dropsand current densities and, thus, the need for evermore robust permeationlayers.

The application of an electric current through the permeation layer hasalso been found to produce considerable chemical and mechanical stresson the thin permeation layer coating at the electrode surface. It hasbeen found that when such thin layers are applied onto electrodeswithout a covalent attachment to the electrode surface, the permeationlayer is prone to separate or ‘delaminate’ from the electrode interface.It is believed this delamination is caused by a change in the chemicalmake-up at the interface between the permeation layer and the electroderesulting from the application of electronic potential at the electrodeand by physical disruption from charged ions and gases emanating fromthe electrode. Thus, the permeation layer must have sufficientmechanical strength and be relatively chemically inert in order towithstand the rigors of changes at the electrode surface withoutinordinate stretching or decomposition.

Thus, the permeation layer of active electronic matrix devices is animportant element in the overall function of the device. It must besufficiently permeable to small aqueous ions, yet efficiently sequesterbiomolecules from the electrode surface. In addition, it must be able towithstand significant chemical and mechanical forces while maintainingits integrity and shape. Several materials have been utilized whichprovide these qualities. Agarose with glyoxal crosslinked streptavidin(SA) has been used as a permeation layer on commercially available,active electronic matrix chips, and the results of electronichybridization of DNA on these chips has been reported in severalpublications (e.g., Sosnowski, et al., Proc. Nat. Acad. Sci. USA,94:1119-1123 (1997), and Radtkey, et al., Nucl. Acids Resrch., 28(7) e17(2000.))

Agarose is a naturally sourced carbohydrate polymer hydrogel, containinglong polymer strands which are crosslinked by non-covalent bonding. Suchhydrogels are referred to as “physical hydrogels”, as they derive theirstructure from non-covalent interactions, as compared to “chemicalhydrogels”, which derive their structure from covalent bonds (orcross-links) between the polymer strands. Agarose permeation layersprovide good relative fluorescent intensity measurements in nucleic acidassays such as hybridization assays for single nucleotide polymorphisms(SNPs) and short tandem repeat sequences (STRs) in amplicon andcapture-sandwich formats, and also in primer-extension type nucleic acidassays which have been used for gene-expression analysis.

However, some disadvantages are encountered in the use of agarose as apermeation layer material. Both the manufacturing process and the factthat agarose is a naturally-sourced product introduce some variation,which may vary performance from batch to batch, necessitating stricterquality controls. This is not ideal for large-scale manufacturing. Thus,an alternative material which is not naturally derived, which can beeasily formed into a permeation layer on the device, and which will meetor exceed the operating standard of agarose, is greatly desirable.

Polyacrylamide and other synthetic polymer gels offer an alternative toagarose hydrogel permeation layers. These materials are whollysynthetic, and thus offer strict quality control of the components. Inaddition, they may be easily molded onto the microelectrode arraysurface with a high degree of uniformity across the entire device.Permeation layers which are between 1 and 2 μm thick in the dry statecan be easily produced in this manner, and are amendable tohigh-throughput manufacture. After molding, streptavidin is covalentlylinked to the surface of the hydrogel to provide attachment sites forbiotinylated oligonucleotide probes or amplicons. Although traditionallyformulated polyacrylamide hydrogels made by the micromolding process areuniform, and offer better product control, they do not perform as wellas the agarose streptavidin permeation layers in most nucleic acidassays. Thus, there is still a need for high-performance syntheticpolymer hydrogel permeation layers for use on active electronic matrixchip devices.

SUMMARY OF THE INVENTION

Surprisingly, applicants discovered that synthetic polymer hydrogelpermeation layers with defined porosity characteristics providehigh-performance characteristics on active electronic matrix chipdevices. Specifically, synthetic polymer hydrogels with mesopores of asize between nanometer-scale nanopores and micrometer-scale microporesperformed as well as or better than the agarose physical hydrogelpermeation layers in various nucleic acid assay formats. Thus, in afirst aspect, the invention provides mesoporous synthetic polymerhydrogel permeation layers for use on active electronic matrix devices.In its most basic format, the assemblage of the permeation layer on thedevice comprises a mesoporous permeation layer overlying an individuallyaddressable electrode on a substrate.

In some preferred embodiments of this aspect of the invention, themesoporous synthetic polymer hydrogel permeation layers comprise poresthat are between 100 and 1000 nm across. More preferably, the permeationlayers comprise pores that are between 100 and 500 nm across, and mostpreferably, the permeation layers comprise pores that are between 200and 500 nm across. As used herein to describe hydrogel pores, “across”is the longest linear dimension across the pore. For some applications,such as those using relatively longer nucleic acids (greater than about70 nucleotides (hereinafter “nt”) in length), primer extension-basedassays, or other enzymatic reactions involving immobilized nucleicacids, it is preferred that the permeation layers also comprisemicropores that are between 1.0 μm and 3.0 μm across, more preferablybetween 1.0 μm and 2.0 μm across. For mesoporous synthetic hydrogelpermeation layer embodiments in which the permeation layer is less than3 μm thick in the dry state, micropores are preferably between 1.0 μmand 1.5 μm across, in order to avoid exposure of the underlyingelectrode.

In other preferred embodiments of this aspect of the invention, themesoporous synthetic hydrogel permeation layers have a porositymeasurement θ of between about 2.0 and about 4.0, wherein θ is definedby the equation;$\theta \equiv \frac{\lambda - \lambda_{0}}{\lambda_{s} - \lambda_{0}} \approx \frac{\lambda}{\lambda_{s}}$wherein integrated light intensity readings λ are taken using dark fieldmicroscopy of a dry hydrogel layer on the test chip (λ), a standardlayer (λ_(S)) with a medium degree of phase separation and a non-phaseseparated, or solid, layer (λ₀) on a Leica darkfield compound opticalmicroscope, and wherein the standard layer is a polyacrylamide hydrogelstandard composition (composition S):

-   -   Acrylamide: Bisacrylamide 19:1 (mol/mol)    -   Total monomer content 20% by weight        The standard layer and the test layer are similarly prepared and        molded (or, optionally, placed) onto the substrate. For the θ        measurement, both the standard permeation layer and the test        permeation layer are about 1.7 μm thick in the dry state. Under        the illumination conditions used in the examples, λ_(S) for the        standard composition was 60±1.5. More preferably, the mesoporous        synthetic hydrogel permeation layers have a θ between about 2.0        and about 3.8. For applications using relatively short nucleic        acids, less than or equal to about 70 nt in length, mesoporous        synthetic hydrogel permeation layers with a θ between about 2.0        and about 3.0 are more preferred. For applications using        relatively longer nucleic acids, greater than about 70 nt in        length, primer extension-based assays, or other enzymatic        reactions involving immobilized nucleic acids, mesoporous        synthetic hydrogel permeation layers with a θ between about 3.0        and about 3.8 are preferred.

Preferred mesoporous synthetic hydrogel permeation layers of theinvention comprise an acryloyl or acrylamido based synthetic polymerhydrogel, more preferably an acrylamide based synthetic polymerhydrogel, and also more preferably a methacrylamide based syntheticpolymer hydrogel. Preferred mesoporous synthetic hydrogel permeationlayers of the invention are also covalently anchored to the underlyingelectrode(s) and/or substrate. In addition, more preferred embodimentsof the mesoporous synthetic hydrogel permeation layers of the inventioncomprise copolymerized attachment moieties for the attachment ofderivatized biomolecules as specific binding entities (e.g., nucleicacids, proteins, etc.). In preferred embodiments, these copolymerizedattachment moieties have the general formula:

wherein,

-   -   P is a polymerizable moiety covalently attached to one or two        moieties selected from the group consisting of: a monomeric unit        of the synthetic polymer and another P-X-R group, as defined        herein, wherein the other P-X-R group may be the same as or        different from the first P-X-R group, further wherein the dashed        line is a covalent bond to the second moiety if P is covalently        attached to two moieties;    -   X is a covalent bond or a linking moiety; and    -   R is a functional moiety for attaching, either covalently or        non-covalently, a biomolecule.

In more preferred embodiments, P is an acryloyl or acrylamido moiety,more preferably an acrylamide moiety. Also, in more preferredembodiments, R is biotin or a biotin binding moiety, such as, forexample, streptavidin or avidin. In other more preferred embodiments, Ris a reactive moiety for use in amine, hydrazine, or hydrazideattachment chemistries. Some preferred R include an active N-hydroxylsuccinimidyl (NHS) ester, a sulfonated NHS ester, an aldehyde, an acid,an acyl halide, a thiol, a disulfide, an amine, an ether, an ester, athioether, a thioester, a hydrazine, or a hydrazide. In some morepreferred embodiments R is a succinimidyl ester. In another group ofpreferred embodiments R is an aldehyde. In yet another group ofpreferred embodiments, R is a hydrazide. In other preferred embodiments,R is a psoralen.

As the use of copolymerized attachment moieties has proven advantageousin order to provide a greater density of attachment moieties for theattachment of specific binding entities (such as nucleic acids orproteins) to synthetic polymer hydrogel permeation layers for activeelectronic matrix chip devices, the synthetic polymer hydrogelpermeation layers with copolymerized attachment moieties of any porosityare another aspect of the present invention. These permeation layersoverlie at least one electrode on a substrate, and comprisecopolymerized attachment moieties of the general formula:

as defined above.

In more preferred embodiments, P is an acryloyl or acrylamido moiety,more preferably an acrylamide moiety. Also, in some preferredembodiments, R is a reactive moiety for use in amine, hydrazine, orhydrazide attachment chemistries, such as an active N-hydroxylsuccinimidyl (NHS) ester, a sulfonated NHS ester, an aldehyde, an acid,or the like. In other preferred embodiments, R is a moiety capable ofparticipating in a biotin-binding pair reaction, i.e., either abiotin-moiety or a biotin-binding moiety. Thus, these preferred Rmoieties in the present invention bind to derivatized biomolecules,which have been derivatized to contain either a biotin-moiety or abiotin-binding moiety (e.g., streptavidin). As many biomolecules may bereadily derivatized with biotin or a biotin analog, in preferredembodiments of the invention R is a biotin-binding moiety such as, forexample, streptavidin, avidin, or chemical or recombinantly engineeredderivatives thereof. As will be appreciated by those of skill in theart, the attachment molecule may contain more than one P moiety attachedto an R moiety, especially in the case of proteinaceous multi-subunit Rmoieties such as streptavidin. In some preferred embodiments of theinvention, the attachment molecule has about one P moiety for eachbiotin-binding site on R. In other preferred embodiments of theinvention, the attachment molecule has about one P moiety for each R.Preferred X may include a covalent bond, as well as commonly utilizedhydrophilic linker molecules such as polyethylene glycols (PEGs), whichmay be connected to R and P by common linkage chemistries such as ester,amide, ether or other common linkage chemistries. Linkage chemistrieswhich are more stable over a wide pH range, such as amide linkages, arepreferred.

In preferred embodiments of the permeation layer aspects of theinvention, the permeation layer is anchored by a covalent linkage to theelectrode, to the substrate, or to the electrode and the substrate. Inthese embodiments, silane-based linkers comprising a copolymerizablemoiety are preferably utilized to covalently anchor the permeationlayer.

Generally, in preferred embodiments of the permeation layers of theinvention, the permeation layers are between about 0.5 μm and about 10μm thick in the dry state, more preferably between about 1.0 μm andabout 5.0 μm thick in the dry state, and most preferably between about1.0 μm and about 2.0 μm thick in the dry state [or at least about 8-9 μmthick when in equilibrium with aqueous buffer]. Also, in preferredembodiments, the permeation layers vary in thickness across theelectrode array of the active electronic matrix chip device by less than0.5 μm, more preferably by less than 0.2 μm, and most preferably by lessthan 0.1 μm, as measured in the dry state.

In another aspect, the present invention provides methods for theproduction of mesoporous synthetic hydrogel permeation layers on activeelectronic matrix chip devises by in-situ polymerization. These methodsgenerally comprise:

-   -   placing an appropriate volume of a polymerization mixture        comprising polymerizable monomers, a cross-linking agent, and a        porogen into a mold cavity of a micromold, wherein the mold        cavity comprises a bottom and at least one side;    -   contacting a substrate, comprising a plurality of electrodes on        the substrate, with the mold to form a closed volume of the        polymerization mixture, wherein the closed volume is in contact        with at least one of the electrodes on the substrate;    -   polymerizing the polymerization mixture; and    -   removing the micromold,        to reveal the polymerized mesostructured synthetic hydrogel        permeation layer overlying at least one electrode on the        substrate. In preferred embodiments, the porogen is a templating        porogen, such as, e.g., a micelle forming porogen, a solid        templating porogen, or a liquid templating porogen. More        preferably, the templating porogen is a micelle forming        surfactant, most preferably a nonionic or zwitterionic        surfactant comprising an aliphatic carbon chain and a polyether,        such as Brij surfactants. In embodiments where a templating        porogen is utilized, the method of the invention preferably        comprises an additional step of selectively removing the        templating porogen with a solvent. In a more preferred        embodiment, the template forming porogen is a micelle forming        surfactant, and the method further comprises selective removing        the surfactant with water.

In preferred embodiments of the method of producing mesoporous synthetichydrogel permeation layers, the polymerization mixture comprisesacryloyl or acrylamido monomers, most preferably an acrylamide monomersuch as methacrylamide. Additionally, in preferred embodiments, thepolymerization mixture comprises a polymerizable cross-linker (forexample, methylene bisacrylamide when the monomer is an acrylamide). Inpreferred embodiments, the polymerization mixture comprises apolymerization free-radical initiator. In more preferred embodiments,the free-radical initiator is a photoinitiator, the micromold bottom istransparent to a radiation of a wavelength which activates thephotoinitiator, and the polymerization step comprises irradiating themold with radiation of the wavelength appropriate to activate thephotoinitiator.

In other aspects of the present invention, the mesoporous synthetichydrogel permeation layer active electronic matrix device of theinvention is electronically addressed with derivatized biomolecules toproduce an addressed mesoporous synthetic hydrogel permeation layerdevice with specific binding entities attached at one or moremicrolocation of the devices. In some preferred embodiments, thebiomolecules are nucleic acids, e.g., nucleic acid probes or samplenucleic acids. In other preferred embodiments the biomolecules arepeptides, polypeptides, or proteins (such as antibodies).

In other aspects of the present invention, the mesoporous synthetichydrogel permeation layer device of the invention is used in a nucleicacid based assay or reaction. Exemplary assay and reaction formatsinclude, but are not limited to: hybridization assay formats (forexample, dot blot assay formats, reverse dot-blot assay formats, nucleicacid sandwich assay formats, base-stacking stabilized hybridizationassay formats) with or without electronic stringency and/or electronicwashing, primer extension reporting assays (for example, gene expressionanalysis with primer extension reporting), immobilized nucleic acidamplification reactions (for example, strand displacement amplification,ligation-dependent strand displacement amplification, polymerase chainreaction amplification, transcription mediated amplification or nucleicacid sequence based amplification, rolling circle amplification),ligation reactions, and nucleic acid cleavage reactions (for example,restriction endonuclease reactions, endonuclease reactions, andexonuclease reactions.) In addition, the mesoporous synthetic polymerhydrogel permeation layer devices of the invention may used in nucleicacid or polypeptide synthesis applications, as has been described forAPEX devices.

In additional aspects of the present invention, the mesoporous synthetichydrogel permeation layer device of the invention is used in animmunochemistry based assay. Exemplary assay formats include, but arenot limited to: addressing the microlocations of the device withantigens (e.g., peptides, proteins, carbohydrates, lipids,proteoglycans, glycoproteins, etc.) in order to assay for antibodies ina bodily fluid sample by sandwich assay, competitive assay, or otherformats; and addressing the microlocations of the device withantibodies, in order to detect antigens in a sample by sandwich assay,competitive assay, or other assay formats.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A photograph of the electronics of a NanoChip® 100 site device,having electrodes with an electrode diameter of 80 μm and center tocenter spacing of 200 μm. The individually controllable electrodesdefine the active sites of the device. The electrodes shown are a layerof platinum (a non-reactive noble metal] formed upon a layer oftitanium/tungsten, which is formed upon a layer of a silicon dioxidesubstrate. Note that the conductive layer of electrodes is overlaid witha layer of insulating silicon dioxide, which is etched away to revealthe electrode pads. Also note the ring of counter electrodes surroundingthe central 10 by 10 array, which can be oppositely biased to allow useof all of the microlocation electrodes in a (+) or (−) bias state.

FIG. 2: A stereomicroscope dark-field photograph of the same type ofdevice, with a mesoporous permeation layer. The permeation layer wasmicro-molded onto the device.

FIG. 3: A reaction scheme showing the formation of two derivatizedstreptavidin (SA) proteins for co-polymerization with acrylamidehydrogels. Reaction of SA with N-acryloxysuccinimide yields acrylamidegroups, while the reaction of SA with theacryloyl-PEG-N-hydroxysuccinimidyl ester yields a SA with PEG-esterlinked acrylate moieities. Ester-linked SA derivatives are lesspreferred for use in hydrogel formulations because of their decreasedstability over a wider range of pH. Similarly, attachment chemistriesare not base- or acid-labile are generally more preferred, but notnecessary, for use in the synthetic polymer hydrogels of the permeationlayer formulations. For instance, N-acryloxysuccinimide may be used asan attachment group for co-polymerization if amine, hydrazide, orsimilar chemistries are to be used to covalently attach, e.g., a nucleicacid probe or amplicon to the permeation layer of the device.

FIG. 4: A reaction scheme illustrating the copolymerization of anacryloyl-PEG-SA derivative with acrylamide in the presence of Brijmicelles to form a mesoporous acrylamide crosslinked hydrogelimpregnated with SA attachment moieties. Note that when thepolymerization takes place above an active electronic array device witha surface which has been activated with a linker silane derivative (suchas Bind Silane™, also 3-methacryloxy-propyl-trimethoxysilane), thepolymer hydrogel matrix becomes covalently linked to the surface of thedevice by incorporation of the linker moiety into the polymer.

FIGS. 5A and B: Scanning electron micrographs of the surface of a moldedphysically homogenous nanoporous polyacrylamide hydrogel permeationlayer (4012-1) on a device. No templating agent (Brij surfactant) wasused in this formulation. The surface of the gel appears relativelysmooth in the 5,000× scan (5A), scale bar 5.00 μm. The surface showsonly nanometer-scale pores in the 45,000× scan (5B), scale bar 500 nm.This is consistent with the estimated pore size in homogeneouslypolymerized acrylamide hydrogels reported elsewhere. These pores, lessthan 100 nm in size, are referred to as “Class I pores” herein.

FIGS. 6A and B: Scanning electron micrographs of the surface of a moldedphysically heterogeneous mesoporous polyacrylamide hydrogel permeationlayer (4012-2) on a device. 11 mg/ml of templating agent (Brij 700surfactant) was used in this formulation. The surface of the gel appearspitted in the 5,000× scan (6A), scale bar 5.00 μm. The surface shows100-500 nanometer-scale pores in the 50,000× scan (6B), scale bar 500nm. These pores, from about 100 nm to about 500 nm in size, are referredto as “Class II pores” herein.

FIGS. 7A and B: Scanning electron micrographs of the surface of a moldedphysically heterogeneous mesoporous/microporous polyacrylamide hydrogelpermeation layer (4012-3) on a device. 18 mg/ml of templating agent(Brij 700 surfactant) was used in this formulation. The surface of thegel appears pitted and craggy in the 5,000× scan (7A), scale bar 5.00μm. Micrometer scale pores are visible at this magnification. Thesepores, from about 500 nm to about 2.00 μm, are referred to as “Class IIIpores” herein. The surface shows class II pores in the 50,000× scan(7B), scale bar 500 nm.

FIGS. 8A and B: Scanning electron micrographs of the surface of a moldedphysically heterogeneous mesoporous/microporous polyacrylamide hydrogelpermeation layer (4006-1) on a device. 73 mg/ml of templating agent(Brij 700 surfactant) was used in this formulation. The surface of thegel appears pitted and craggy in the 5,000× scan (8A), scale bar 5.00μm, displaying class III pores. The surface shows class II pores in the50,000× scan (8B), scale bar 500 nm.

FIG. 9: A darkfield compound microscope photograph (Leica iNM 100 withan Optromics™ video camera) of a single microlocation, or pad, of adevice, like those used to determine the light scattering values for theθ calculation. The pad diameter is 80 μm.

FIG. 10: A chart showing θ measurements for several batches of 4006-1permeation layers formed on NanoChip® devices. The θ porositymeasurement is very consistent over twenty different batches (mean 3.63θ, std±0.07 θ). These data show that mesoporous synthetic hydrogelpermeation layers can be consistently molded onto active electronicmatrix devices. Likewise, the consistency of the light-scatteringmeasurement technique is also validated.

FIG. 11: A chart showing mean fluorescent intensity (MFI) readings forthe SNP1 assay described in Example 4, performed on three activeelectronic matrix chip device with 4006-1 permeation layers, and theresults of the same assay performed on a standard SA-agarose permeationlayer chip. Note that the fluorescent intensity readings on the 4006-1chips are generally higher than those on the SA-agarose chip, and thatboth allele probes (C-allele—Cy-3, and T-allele—Cy-5) give goodreadings, allowing the easy identification of heterozygous ampliconmixtures.

FIG. 12: A chart showing MFI readings from the two nucleic acidimmobilization experiments described in Example 5. The 46 nt oligomersimmobilization experiment results are indicated by diamonds (♦), and the114 nt amplicon immobilization experiment results are indicated bysquares (▪). Note that the relative binding efficiency of the largernucleic acid increases significantly with increased porosity, while thebinding efficiency of the small nucleic acid decreases very slightlywith increased porosity.

FIG. 13: A chart showing MFI readings from primer extension assayexperiments described in Example 6, in which sample amplicons of 0.5 nM,1.0 nM, 2.0 nM, 4.0 nM, 8.0 nM and 16.0 nM concentrations were addressedto multiple sites on chips with 4012-1, 4012-2, 4012-3, or 4006-1permeation layer formulations. Note that the fluorescent intensity doesnot increase in a linear fashion with increasing nucleic acidconcentration on the nanoporous permeation layer chip, 4012-1 (♦), butdoes increase in a linear fashion on the mesoporous permeation layerchips 4012-2 (▴), 4012-3 (▪), and 4006-1 ( ).

FIG. 14: A chart showing θ measurements for synthetic polymer hydrogelpermeation layers made by using various amounts of Brij 700 surfactantas a template porogen.

FIG. 15: A schematic drawing of the micromolding process. Starting witha micromold with a bottom transparent to a light wavelength (hv), a dropof polymerization mixture is placed into the mold in step 1. In step 2,the active electronic matrix chip substrate is brought into contact withthe mixture and the mold, forming a closed volume of the polymerizationmixture. In step 3, the mixture is polymerized by irradiating the moldwith a light wavelength appropriate to initiate polymerization. In step4, the substrate is removed, along with the newly formed permeationlayer over the electrodes, allowing the process to start over again witha new active electronic matrix chip substrate in step 5.

DETAILED DESCRIPTION OF THE INVENTION

As has been described, a key role in the function of active electronicmatrix devices is played by the ion-permeable permeation layer whichoverlies the electrodes of the microlocations, or active sites, of thesedevices. As part of its function, the permeation layer providesattachment moieties for the attachment and immobilization of nucleicacids (or other specific binding entities, such as antibodies, orsynthetic binding moieties such as pyranosyl-RNA). More importantly, thepermeation layer separates the attached or tethered oligonucleotides andhybridized target DNA sequences from the highly reactive electrochemicalenvironment generated immediately at the electrode surface. This highlyreactive electrode surface, and the electrochemical productsconcentrated at the electrode surface, can rapidly destroy DNA probesand target DNA sequences which contact the surface or approach it tooclosely. Similar detrimental effects may be encountered with othermacromolecular binding entities immobilized directly on the electrodesurface. The permeation layer allows oligonucleotides and DNA fragmentsto be electronically concentrated above, rather than on, the electrodesurface and hybridized to anchored complementary oligonucleotides whilebeing protected from the reactive electrode surface and its immediateenvironment. The permeation layer also allows the gradual diffusion ofthe electrochemical reaction products (H⁺, OH⁻, gasses, etc.) into thesolution around the microlocation, allowing these products to balancethe charge through the permeation layer by ion exchange and to reactwith buffer species. Thus, the design of the microelectrode andpermeation layer, forming a microlocation structure, allows high currentdensities to be achieved in a very confined area, while minimizing theadverse effects produced by the electrode itself.

Once specific binding entities, such as nucleic acids, have beenaddressed to microlocations and immobilized, the addressed devices areable to control and actively carry out a variety of assays andreactions. Analytes or reactants can be transported by free fieldelectro-phoresis to any specific microlocation where the analytes orreactants are effectively concentrated and reacted with the specificbinding entity at the microlocation. The sensitivity for detecting aspecific analyte or reactant in dilute sample solutions is improvedbecause of this concentrating effect. An additional advantage, whichalso improved the specificity of the assays carried out on the device,is that any un-bound analytes or reactants can be removed by reversingthe polarity of a microlocation (also known as “electronic washing”.)

The ability to produce a precisely controlled high current level, ordensity, at individual microlocations even allows the selective“de-hybridization” of DNA fragments, achieving hybridization selectivityat the level of single base mismatches. Thus, the devices can furtherimprove the specificity of assays and reactions by providing anotherparameter to encourage mismatch de-hybridization (along with the moretraditional parameters of temperature and chemical environment), whichis known as “electronic stringency”, or “electronic stringency control(ESC).” For DNA hybridization reactions which require differentstringency conditions, ESC overcomes an inherent limitation ofconventional array technologies, which must rely on stringencyconditions which are consistent for all sites over the entire array. Theactive devices of this invention can electronically produce differentstringency conditions at each microlocation. This adds anothercontrollable factor affecting hybridization, along with the moretraditional factors such as temperature, salt concentration and thepresence of chaotropic agents. Thus, all hybridizations can be carriedout optimally in the same bulk solution, and multiple hybridizationreactions can be carried out with minimal outside physicalmanipulations. Additionally, it may be unnecessary to change temperaturein some cases, and the need for multiple washing procedures is greatlyreduced.

Thus, the permeation layer of active electronic matrix devices is morethan simply a mechanical support to hold attachment sites for specificbinding entities. It is also an important factor in the overallperformance and efficiency of the devices in their active electronicmodes. Unlike coatings or gel supports which have been described forpassive array devices, e.g., the gel-block arrays described in U.S. Pat.No. 5,770,721, which simply use hydrogel matrices as an attachmentscaffold, permeation layers used on the active electronic matrix devicesdescribed herein must also allow the efficient active electronictransport of biomolecules to the microlocations of the device, and beconducive to electronic hybridization and/or stringency procedures.

As noted above, agarose hydrogels containing glyoxal-crosslinkedstreptavidin have proven to be effective permeation layer materials onactive electronic matrix chip devices. In general, these permeationlayer formulations have provided good mean fluorescence indices withminimal background. SNP assays run on the SA-agarose chips havedemonstrated nearly 100% accuracy in several tests run with actualgenomic samples, together with a high discrimination ratio fordiscerning between alleles in both homozygous and heterozygous samples.In addition, very good results have been obtained using SA-agaroseactive electronic matrix chips in STR and gene expression analysisassays.

However, as also described above, the use of SA-agarose as a permeationlayer has several disadvantages in the manufacturing context. Agarose isa physical hydrogel, which derives its semi-solid structure fromnon-covalent interactions between long polysaccharide chains. As theseinteractions are temperature-dependent, changes in temperature changethe viscosity of the agarose solution: at higher temperatures, thesolution is more liquid, while it forms a solidified gel at roomtemperature. Thus, in order to coat the agarose permeation layer ontothe active electronic matrix chip electrode array, the agarose solutionmust be kept at a relatively high and constant temperature during themanufacturing process. This also must be balanced with maintaining theactivity of the streptavidin crosslinked to the agarose in the solution,which can denature if the temperature is too high. The currentmanufacturing method is to spin-coat the agarose solution onto theactive electronic matrix chip surface. Thus, the agarose permeationlayer production methods add significantly to the resources expended inproducing the device.

Although this produces a fairly uniform thickness, sub-micron variationsin thickness are often encountered when comparing the thickness of thepermeation layer over microlocations on different sites on the chip. Inaddition, because agarose is a natural product, batch to batchvariability may be seen with regard to its chemical characteristics andits performance as a permeation layer. This variability in both thematerials and the manufacturing methods decreases the number of activeelectronic matrix chips which will meet quality control standards, alsoincreasing the resources necessary to produce high-quality activeelectronic matrix chips with agarose-based permeation layers.

In contrast to the naturally-sourced physical hydrogels, such asagarose, synthetic polymer chemical hydrogels offer a more easilycontrolled quality and production characteristics. Synthetic polymerhydrogels are produced from individual monomeric components, which areusually synthesized themselves from basic organic chemical components.The monomers can be purified to very high quality, with identicalphysical and chemical characteristics between production batches. Themonomeric components can be mixed in various formulas with cross-linkermoieties and polymerized by a triggered initiator (e.g., by exposure ofa photoinitiator to UV). Thus, chemical hydrogels offer strict controlover the rate of polymerization and the characteristics of the resultinghydrogel, as compared the control afforded by physical hydrogels formedby pre-polymerized chains.

In addition, synthetic polymer hydrogels offer many advantages for massproduction. They be easily molded onto the microelectrode array surfacein situ with a high degree of uniformity across the entire device.Microreaction molds and methods of using them to form thin, uniform,synthetic polymer hydrogel layers on the surface of active electronicmatrix chips have been described in WO 01/43938, Havens et al,incorporated herein by reference in its entirety. The microreactionmolds disclosed comprise a mold cavity, with at least one sidetransparent to an electromagnetic radiation wavelength. In thesesystems, a small volume of the polymerization mixture (monomers,cross-linkers, and photoactivator) is placed into the mold cavity. Themicroelectrode array substrate is then pressed against the mold, formingan enclosed volume of the polymerization mixture on the substrate. Thepolymerization reaction is initiated by irradiating the enclosed volumewith an appropriate wavelength of light for the photoinitiator (e.g.,UV), and the polymerization reaction is allowed to proceed tocompletion. When the mold is removed, a thin, uniform, synthetic polymerhydrogel permeation layer has been formed on the microelectrode array.

Permeation layers which are between 1 and 2 μm thick, with sub-micronvariations in thickness, can be easily produced in this manner, and areamendable to high-throughput manufacture. Multi-layer permeation layers(either overlaid or graft-polymerized onto the prior layers) be made inthis manner as well, by using a series of molds with differing depthsand/or widths. In addition, the molds can be designed to form individualpermeation layers over each individual microelectrode, creatingindividually formed microlocations. In this manner, it is even possibleto vary the permeation layer composition from microlocation tomicrolocation over the array of the active electronic matrix chip.

Given the advantages of using synthetic polymer hydrogel permeationlayers, various synthetic polymer hydrogel formulations were tested foruse as permeation layer materials. Initially, polyacrylamide gels whichwere surface derivatized with SA were tested. However, the standardnanoporous polyacrylamide formulations used did not produce permeationlayers which provided fluorescent intensity, signal linearity, and otherperformance characteristics which were comparable to those obtainedusing agarose-SA physical hydrogel permeation layers. Thus, alternativeformulations were explored which would alter the physicalcharacteristics of the polyacrylamide chemical hydrogel matrix.

Surprisingly, it was discovered that polyacrylamide synthetic polymerhydrogel permeation layers with defined porosity characteristicsperformed dramatically better in all of the nucleic acid assay formatstested, as compared to synthetic polymer hydrogel permeation layers withpore sizes smaller than the defined range. These permeation layerspossess pores in a middle size range: between about 100 and about 1000nm across. Thus, these synthetic polymer hydrogel permeation layers havebeen designated “mesoporous,” as opposed to possessing nanometer-scalepores (“nanoporous”), or micrometer-scale pores (“microporous”).Mesoporous synthetic hydrogel permeation layers also show a macroscopicdifference from nanoporous permeation layers, as shown in FIG. 2.Instead of a clear gel, mesoporous synthetic hydrogel permeation layersappear milky or translucent, due to the scattering of light through thelayer caused by the separation of the gel and solution phases. Thislight-scattering effect, also shown in the dark field micrograph in FIG.9, forms the basis for the porosity measurement 0, discussed below andin Example 3.

The characteristics of increased porosity in synthetic polymer hydrogelformulations were further characterized using a template-type porogen tointroduce various degrees of porosity, or phase separation, into thesynthetic polymer hydrogels. As the hydrogel comprises a meshwork ofpolymer strands in the aqueous solution, it can be thought of as atwo-phase system: the meshwork, or solid phase, and the surroundingsolution phase. When the meshwork is separated to create pores (or areaswhere the meshwork is not), then the phases become more separate,creating larger and more distinct pore/void areas. By using amicelle-producing surfactant, Brij 700, the degree of phase separationcould be controlled by varying the concentration of the surfactant inthe polymerization solution, thus varying the prevalence and averagesize of the micelles.

As described in Example 1, four permeation layer formulations, withdifferent degrees of phase separation, were used to produce activeelectronic matrix chip devices which were the subject of the majority ofexperiments: 4012-1, with 0 mg/ml Brij 700 [No porogen—a basicacrylamide/bisacrylamide chemical hydrogel with copolymerizedstreptavidin attachment moieties]; 4012-2, with 11 mg/ml Brij 700;4012-3, with 18 mg/ml Brij 700; and 4006-1, with 73 mg/ml Brij 700.4012-2, 4012-3, and 4006-1 are all considered to be mesoporous syntheticpolymer hydrogel permeation layers. Electron micrographs (FIGS. 5A & B,6A & B, 7A & B, and 8A & B) were taken of the permeation layers,prepared as described in Example 2. As can be clearly seen from the45,000× and 50,000× scans of the permeation layers, 4012-1 appears to berelatively smooth, with pores in the nanometer range. In contrast theother three formulations have a fairly consistent fine morphology atthis magnification, showing mesopores between about 100 and about 500 nmacross. In addition, in the 5,000× scans, 4012-3 and 4006-1 also exhibita microporous morphology, with pores between about 1 μm and about 2 μmacross.

In order to more easily quantify the porosity characteristics of thepermeation layer formulations, the measurement θ was devised, asdescribed in Example 3, based on light-scattering analysis under darkfield microscopy. The standard layer is a polyacrylamide hydrogelstandard composition (composition S):

Acrylamide: Bisacrylamide 19:1 (mol/mol)

Total monomer content 20% by weight

Under the illumination conditions used in the examples, λ_(S) for thestandard composition was 60±1.5. By comparison to the standardcomposition S, the relative phase separation, and thus porosity, of thesynthetic polymer hydrogel permeation layers can be quantified. The θ of4012-1 was 1.5, while the θ's of the mesoporous permeation layers werebetween 3.0 and 3.5. In order to more thoroughly characterize the effectof different concentrations of Brij surfactant on the porositymeasurement θ, eight permeation layers based on the 4012-1 formulation,with varying concentrations of Brij 700 surfactant from 3.9 to 170.4mg/ml, as described in Example 7, were compared to the 4012-1formulation. The θ values for these synthetic polymer hydrogelpermeation layers are shown in FIG. 14. As can be seen in the Figure,the porosity of the permeation layers changes rapidly between the 9.3mg/ml formulation (slightly less than 4012-2) with a θ of 2, and the18.6 mg/ml formulation (slightly more than 4012-3) with a theta of about3. This suggests a critical phase separation change for the use of theBrij surfactant template porogen at around 10 mg/ml: above thisconcentration, the θ values hover in the 3.0 to 3.5 range. Althoughapplicants are not bound by any particular theory, one possibility isthat the Brij micelles aggregate at the higher concentrations. Thiscould explain the appearance of micropores in the 4012-3 electronmicrographs. Interestingly, the use of a different streptavidin moietyfor copolymerization produced increased porosity, as measured by θ, asdescribed in Example 3. As seen in FIG. 10, batches of permeation layerswith the same formulation as 4006-1, except for the substitution ofamide-linked acrylamido SA for ester-PEG linked acryloyl SA (see FIG. 3for comparison) produced mesoporous synthetic hydrogel permeation layerswith θ's averaging about 3.6. Thus, the porosity of the mesoporoussynthetic hydrogel permeation layers may be influenced by a combinationof factors, including the presence of porogens, the type ofcopolymerized attachment moiety used, and more conventional parameterssuch as crosslinker ratio and total monomer concentration.

In several nucleic acid assays using electronic addressing andhybridization techniques, these permeation layers showed distinctdifferences in function. As described in Example 5, and shown in FIG.12, the ability of a relatively large polynucleotide (114-mer) to beimmobilized by a biotin-streptavidin interaction in the mesoporoussynthetic hydrogel permeation layers was increased in comparison to thenanoporous permeation layer formulation (4012-1). Conversely, theability of a relatively small (46-mer) oligonucleotide to be immobilizedby a biotin-streptavidin interaction was sufficiently high in thenanoporous permeation layer, and actually decreased slightly in themesoporous permeation layer. Although the invention is not bound by anyparticular theory, these results suggest that, perhaps, its increasedrate of migration through the increased porosity layers, underelectronic addressing conditions, decreased the opportunities for thebiotinylated 46-mer to interact with the streptavidin attachmentmoieties. This hypothesis was strengthened by experiments incubating thefour permeation layers with the 46-mer under passive conditions, underwhich no difference in immobilization was seen. Similar results weredemonstrated with a wider range of range of porogen concentrationformulations in the experiments described in Example 7, and shown inFIGS. 15 and 16.

In a separate set of experiments, the four permeation layers were usedin a primer-extension reporting assay format as described in Example 6.These experiments demonstrate the ability of the mesoporous synthetichydrogel permeation layers to exhibit linearly increased hybridizationand detection by enzymatic primer extension reporting using a moderatelysized oligonucleotide (about 80 nt long, a typical size of those used ingene expression assays). As shown in FIG. 13, the fluorescent intensityon the mesoporous synthetic hydrogel permeation layers approximatelydoubled for every two fold increase oligonucleotide concentration over awide concentration range, while it flattens out quickly on thenanoporous 4012-1 permeation layer. These results indicate that themesoporous synthetic hydrogel permeation layer formulations provideimproved performance for applications in which quantitative analysis isused, such as gene expression monitoring and viral load or pathogenconcentration determinations.

The performance of 4006-1 formulation permeation layers on activeelectronic matrix chip devices was compared to a standard agarose-SAchip (MSP) in a SNP-type base-staking reporting assay (SNP 1) asdescribed in Example 4. As can be seen from the data in FIG. 11, themean fluorescent intensity is very strong on the 4006-1 permeationlayers, and is comparable to the MFI readings on the SA-agarose chip. Inaddition, all allelic probes show a strong signal, allowing cleardiscrimination between homozygous and heterozygous samples. Theperformance of this permeation layer formulation was further testedusing seven other SNPs. As shown by the chart in Example 4, themesoporous synthetic hydrogel permeation layer formulation 4006-1provided excellent discrimination ratios between the signals for theallelic probes, and correctly identified all SNPs tested using amplifiedsamples.

As these experiments were based on typical types of nucleic acid assaysutilizing active electronic matrix chip devices, they have directimplications for use of the mesoporous synthetic hydrogel permeationlayer devices of the present invention in similar nucleic acidhybridization-based assays and enzyme reaction or DNA-proteininteraction based assays. As demonstrated by experimental data,mesoporous synthetic hydrogel permeation layers provide surprisinglyimproved detection performance and dynamic range, as compared tonanoporous standard synthetic polymer hydrogel formulations, making themsuitable for a wide range of electronically assisted nucleic acid assayformats. As synthetic binding system applications such as pyranosyl-RNAencoding use similarly sized nucleic acid-like oligomers, althoughcreating different secondary structures, the experimental results alsoindicate that the mesoporous synthetic hydrogel permeation layers willoffer improved device performance in applications using syntheticbinding systems. In addition, the performance of the new mesoporoussynthetic hydrogel permeation layer devices in nucleic acid assays,especially the enzymatic reaction assays, indicate that they will alsoexhibit improved performance in protein-based applications such asimmunoassays.

Porosity Control in Synthetic Polymer Hydrogel Permeation Layers

As described above, the improved characteristics of the mesoporoussynthetic hydrogel permeation layer devices of the invention depend uponthe presence of mesopores in the synthetic polymer hydrogel permeationlayer on the devices. Thus, a key step in the production of themesoporous synthetic hydrogel permeation layer is the control ofporosity in the polymerizing hydrogel matrix, when the monomers andcross-linkers are polymerized into a polymer network. Practically,various porosities can be accomplished in two ways: a) producing arelatively physically homogeneous hydrogel matrix structure in which thespaces between the polymer matrix are of a sufficient distance toproduce pores of the desired size, and b) producing a relativelyphysically heterogeneous synthetic polymer hydrogel matrix structure byencouraging the developing polymer strands to cluster into dense areasand void areas, usually by forming the matrix around a removabletemplate structure of an appropriate size. Either strategy may beutilized alone, or in combination with the other, to produce themesoporous synthetic hydrogel permeation layer devices of the invention.

Physically Homogeneous Methods: Polymerization Control

Synthetic polymer hydrogels are porous to a greater or lesser extent bynature, consisting primarily of a random network of cross-linked polymerstrands, and an aqueous solution filling the spaces between the strands.This hydrated network creates a semi-solid structure, which has porositycharacteristics primarily determined by the conditions ofpolymerization, including: total concentration of monomers (e.g.,individual monomer moieties or block co-polymer units) and anycross-linker molecules in the polymerization mixture, the relativepercentage of cross-linking agent (often a molecule with two or morepolymerizable groups) in the polymerization mixture, and the rate ofpolymerization (which can be effected by the type, concentration, andactivation of an initiator molecule, the temperature of thepolymerization reaction, and other known parameters).

The type of synthetic polymer hydrogel utilized in the mesoporoussynthetic polymer hydrogel permeation layers of the invention is notintended to be limited to acrylamide-chemistry hydrogels, althoughacrylamide/bisacrylamide systems are utilized as an exemplary syntheticpolymer hydrogel system in the examples. In general, any sufficientlyhydrophilic and polymerizable molecule may be utilized in the productionof a synthetic polymer hydrogel for use as a permeation layer.Polymerizable moieties in the monomers may include alkenyl moietiesincluding but not limited to substituted or unsubstitutedα,β,unsaturated carbonyls wherein the double bond is directly attachedto a carbon which is double bonded to an oxygen and single bonded toanother oxygen, nitrogen, sulfur, halogen, or carbon; vinyl, wherein thedouble bond is singly bonded to an oxygen, nitrogen, halogen, phosphorusor sulfur; allyl, wherein the double bond is singly bonded to a carbonwhich is bonded to an oxygen, nitrogen, halogen, phosphorus or sulfur;homoallyl, wherein the double bond is singly bonded to a carbon which issingly bonded to another carbon which is then singly bonded to anoxygen, nitrogen, halogen, phosphorus or sulfur; alkynyl moietieswherein a triple bond exists between two carbon atoms.

Acryloyl or acrylamido monomers such as acrylates, methacrylates,acrylamides, methacrylamides, etc., are advantageous because of thelarge body of knowledge which has been accumulated concerning theformulation of hydrogels using these polymers. More preferred acrylamidomonomers include acrylamides, N-substituted acrylamides, N-substitutedmethacrylamides, and methacrylamide. However, other polymers are alsouseful, such as epoxide-based polymers, vinyl-based polymers,allyl-based polymers, homoallyl-based polymers, cyclic anhydride-basedpolymers, ester-based polymers, ether-based polymers, alkylene-glycolbased polymers (e.g., polypropylene glycol), and the like. Thus, themesoporous synthetic polymer hydrogel permeation layers of the inventionencompass numerous synthetic polymer hydrogel compositions, which may befacilely devised by those of skill in the polymer arts. The primaryconsiderations in selecting a synthetic polymer hydrogel are sufficientmechanical strength for use as a permeation layer, sufficienthydrophilicity to ensure sufficient solution volume in the gel, very lownon-specific binding of biomolecules such as nucleic acids to thepolymer hydrogel, and the adaptability of the synthetic polymer hydrogelto various methods for the production of mesoporous structures in thehydrogel matrix.

In preparing the polymerization mixture, it is possible to includewithin the permeation layer substances which can reduce the adversephysical and chemical effects of electrolysis reactions, including, butnot limited to, redox reaction trapping substances (e.g., palladium forH₂, and iron complexes for 0₂ and peroxides). A sub-layer of thepermeation layer may be designed for this purpose. Additionally, thepermeation layer can contain compounds or materials which help maintainthe stability of the DNA hybrids; these can include but are not limitedto histidine, histidine peptides, polyhistidine, lysine, lysinepeptides, and other cationic compounds or substances.

For polyacrylamide based hydrogels, the relationship between the factorsof monomer concentration, cross-linker percentage, polymerizationconditions and porosity has been studied in the context of gelformulations for electrophoretic separations through the gel matrix, andone of skill in the art will appreciate the ability to manipulate therelative porosity of a polyacrylamide hydrogel using these parameters.In general, lowering the concentration of the total monomerconcentration in the polymerization mixture will increase the porosityof the resulting hydrogel. However, lowering the concentration creates aless solid polyacrylamide hydrogel which lacks sufficient mechanicalcohesion, and the maximum pore size obtainable through this methodappears to be about 100 nm. Alternatively, the percentage ofcross-linking agent can be increased to increase the porosity of theresulting gel. But, increasing cross-linker concentration produces aless hydrophilic polymer network, which eventually collapses andexcludes the aqueous solution at higher cross-linker concentrations(e.g., about 30% for methylene bisacrylamide and about 50% for DHEBA).Even at these maximum workable concentrations, pore size may only beincreased to about 200-300 nm. In addition, highly crosslinkedpolyacrylamide gels become increasingly friable, which also reduces themechanical robustness of these synthetic polymer hydrogel formulationsfor use as a permeation layer. The use of different solvent systems hasalso been demonstrated to alter the porosity characteristics ofsynthetic polymer hydrogels. For example, the introduction of a lesspolar co-solvent, such as DMSO, may be used to form more porous networksof crosslinked polymer chains in polyacrylamide matrices.

In addition, applicants have discovered that the use of differentcopolymerizable attachment moieties (discussed in further detail below)can have a significant impact on the overall porosity of the resultingsynthetic polymer hydrogel. For instance, in Example 3, switching from aSA-PEG-acryloyl molecule to an SA-N-acrylamide molecule increase theporosity of the 4006-1 formulation from a θ of about 3.0 to about 3.6.Thus, changes in the copolymerized attachment molecule in thepolymerization mixture may also be utilized to increase the porosity ofthe resulting synthetic polymer hydrogel.

Thus, several techniques involving the basic formulation of thepolymerization mixture have been exploited to increase polyacrylamidehydrogel porosity. However, even combinations of these techniques areonly sufficient to generate pores in polyacrylamide hydrogels which areat the lower end of the mesopore size range. In addition,low-concentration polyacrylamide hydrogels with high cross-linkerconcentrations tend to be mechanically weak and friable, and can haveundesirable hydrophobic characters. These materials may still be used asmesoporous synthetic hydrogel permeation layer compositions. But, foruse as permeation layers, it is preferred that a physicallyheterogeneous polyacrylamide hydrogel be used, which may be formed bythe methods described below.

It will, however, be appreciated by those of ordinary skill in the artthat these mechanical limitations apply primarily to polyacrylamidehydrogels. The general principles of increasing porosity by decreasingmonomer concentration and increasing cross-linker concentration may alsobe utilized to alter the porosity characteristics of other polymerhydrogels with fewer adverse mechanical strength effects. Thus, if anon-acrylamide polymer with a greater intrinsic mechanical strength isutilized, one may devise a physically homogeneous mesoporous synthetichydrogel permeation layer composition which will have the desiredmechanical strength.

Physically Heterogeneous Methods: Templating and Other Strategies

Because of the difficulties in obtaining a mechanically strongmesoporous synthetic hydrogel permeation layer formulation usingphysically homogeneous methods with acrylamide based synthetic polymerhydrogel, it is preferred that a physically heterogeneous structurestrategy be utilized with this polymer material. In addition, because ofthe added control over pore size and distribution characteristics, thesestrategies are generally preferred to produce mesoporous synthetichydrogel permeation layers for use in the present invention. Severaltools for creating void spaces in synthetic polymer hydrogel matriceshave been described in the context of electrophoretic separationmaterials and gel chromatography materials, including: the use oftemplate porogens (e.g., micelles forming agents, solid mesobeads,liquid emulsions); the use of lateral aggregation of the polymer, andother methods such as bi-phasic copolymerizable systems.

In general, the use of templating porogens has allowed the greatestflexibility in producing synthetic polymer hydrogels with defined poresizes. The basic process is simple: 1) a template of the desired size ismixed into the polymerization mixture; 2) the mixture is thenpolymerized, forming the network of polymerized cross-linked monomersaround the template; 3) after polymerization, the template isselectively removed with a solvent solution, leaving behind thesynthetic polymer hydrogel with the desired mesoporous structure. Thus,the primary consideration in choosing a templating porogen agent is theability to selectively dissolve the agent in a solvent which will notsignificantly affect or alter the synthetic polymer hydrogel during theleaching process.

Several micelle-forming molecules are ideal for use as template porogensbecause they form spheroid structures with a fairly tight sizedistribution, and are usually extractable with relatively mild solventconditions. The polymerization solution forms a two phase system withthe micelle-forming templating agent: an “external” phase containing thepolymerizable monomers and cross-linkers, and “internal” usuallyconsisting primarily of the micelle forming materials. Compared to asimple immiscible emulsion system, in which the size of thenon-polymerizing phase is less controlled or dependant on surfactantconcentration, micelles are an ordered liquid structure with moredefinite size characteristics. Surfactants are particularly preferred asmicelle-forming template porogens for use in producing mesoporoussynthetic hydrogel permeation layers for use in the invention. Byselecting the proper surfactant molecule, the desired micelle size rangemay be produced. For instance, nano-sized micelles are easily producedusing sodium dodecyl sulfate (SDS), while meso-sized micelles may beproduced using commercially available polymer-based nonionic surfactantssuch as the Brij series. Brij surfactants contain an aliphatic carbonchain and a polyether chain. These are versatile nonionic surfactantswhich can be used to create micelles of various sizes and sizedistributions. Brij 700 is utilized as an exemplary surfactant templateporogen herein, but one of ordinary skill in the chemical arts wouldreadily be able to utilize alternative micelle-forming template porogensin a similar manner.

Surfactant based template porogens have two other advantages, which areof great use in the manufacture of mesoporous synthetic hydrogelpermeation layers on active electronic matrix devices. First, the sizeand number of pores produced, and thus the porosity characteristics ofthe synthetic polymer hydrogel, can be altered by changing theconcentration of the surfactant template porogen in the polymerizationmixture. As shown in FIG. 14, the porosity of a polyacrylamide syntheticpolymer hydrogel may be altered by ˜1.5 θ by modestly increasing amountsof surfactant. This ability to alter the θ value of the syntheticpolymer hydrogel over a significant range is useful for the introductionof micropores into the mesoporous structure, which are preferred inmesoporous synthetic hydrogel permeation layers which will be used inlarge (over 100 nt) amplicon hybridization applications, or with enzymereaction based applications.

Second, surfactant based template porogens are moderately soluble inwater, allowing plain distilled water or aqueous buffer solutions to beutilized in leaching out the template after polymerization, as in theexemplar permeation layers. By avoiding the use of organic solvents ormore chemically reactive compositions, the mechanical strength andmesostructural characteristics of the synthetic polymer hydrogel may bepreserved. In contrast, non-water-soluble templating porogen agentsusually require harsher leaching conditions, which can alter thestructural integrity of the end-product permeation layer.

Instead of surfactants or other micelle forming templating porogens, asolid bead type templating porogen could be utilized. Sub-micronmeso-size glass beads (“mesobeads”) have been described which would makesuitable porogen templates. These types of template porogens have theadvantage of strict control over template size and size distribution, asthis is a stable physical parameter of the solid beads, and the beadsmay be sorted by size prior to inclusion in the polymerization mixture.However, glass beads require leaching from the synthetic polymerhydrogel mesostructure by dissolution with hydrofluoric acid. Inpolyacrylamide gels, this process has not demonstrated an observableadverse effect on the gel structure. However, silicon dioxide layers arecurrently used in the substrate materials of active electronic matrixchip devices, and the exposure of these components to hydrofluoric acidcould adversely affect the electrode array. Thus, the use of glassmesobeads is not a currently preferred method for the production ofmesoporous synthetic polymer hydrogel permeation layer devices of theinvention. However, if alternate non-etchable materials are utilized forthe insulating layer (such as silicon nitride), or a non-silicon basedsubstrate is used for the active electronic matrix electrode array, thedisadvantages associated with the hydrofluoric acid leaching processwould not be a concern.

Another alternate template porogen strategy is the use of immiscibleliquids to form stable (or semi-stable) emulsions with thepolymerization mixture. As many of the polymerization components(monomers, cross-linkers, copolymerizable attachment molecules) aresoluble in an aqueous phase, it is popular to utilize ahydrophobic/hydrophilic two phase system, in which the hydrophobic phaseforms droplets of the desired pore size in the hydrophilic phase.Usually, these emulsions are stabilized with surfactants to preventaggregation and separation of the hydrophobic phase from the hydrophilicphase. Although this method is suitable for use in producing mesoporoussynthetic polymer hydrogel permeation layers, it is not as preferred asthe micelle-forming template porogen method, as the microemulsiondroplets are not as ordered and well defined as the structure ofsurfactant micelles.

A similar strategy is the use of gas-phase porogen templates. Severalporogens are known in the polymer arts which are useful in theproduction of gas bubbles during the polymerization reaction in a welldistributed and controlled manner. The use of gas-producing porogen maybe suitable for especially viscous polymerization mixtures in which thegas bubbles formed would not aggregate to a significant extent withinthe solidifying synthetic polymer hydrogel matrix. However, this methodis not preferred for use with polyacrylamide hydrogels, as they are toofluid.

Non-templating strategies may also be utilized in order to introducevoid spaces into the final synthetic polymer hydrogel matrix in order tocreate a physically heterogeneous mesostructure. For instance, aphenomenon called lateral aggregation has been described, wherein whenpolyethylene glycols of certain molecular weights are included inacrylamide polymerization mixtures, the growing polymer chains aggregatebetween the polyethylene glycol molecules. For instance, when PEGs inthe range of 10,000 to 20,000 MW are added to the polymerization mixtureat concentrations of about 2.0 to 2.5% wt/vol, pores of an average sizeof 500 nm may be produced (see, e.g., Righetti, et al., J.Chromatography, 638:165-178 (1993), incorporated fully herein byreference). Thus, the addition of high molecular weight PEGs may also beutilized to produce mesoporous synthetic polymer hydrogel permeationlayers, especially those based on polyacrylamide hydrogels.

In addition, a controlled polymer network degradation technique has alsobeen described. In this technique, a polyacrylamide matrix was degradedin a controlled manner by periodate oxidation in order to openmicropores in the gel. However, this method would probably not bepreferred for use on the devices of the invention, as the harsh chemicalconditions may be detrimental to the electronics of the device or theattachment moiety chemistries.

As is evident from the above discussion, numerous methods exist toproduce synthetic polymer hydrogel matrices with the desired mesoporous(and, optionally, microporous) character for use in the permeationlayers of the invention. Thus, one of ordinary skill in the polymer artswill appreciate that the mesoporous synthetic polymer hydrogelpermeation layers of the invention are not necessarily limited to layersproduced by a particular method (e.g., the use template porogens), butrather encompass a broad class of mesostructured synthetic polymerhydrogel permeation layers which may be manufactured from a variety ofsynthetic polymer hydrogel materials by a variety of methods.

Synthetic Polymer Hydrogel Formulations With Copolymerization ofAttachment Moieties

Preferred synthetic polymer hydrogels for use in invention containcopolymerized attachment moieties for the attachment of specific bindingentity molecules (e.g., nucleic acids, proteins, polypeptides, syntheticbinding system components such as pyranosyl RNA). Such copolymerizedattachment moieties can generally be described by general formula:

wherein,

-   -   P is at least one polymerizable moiety covalently attached to        one or two moieties selected from the group consisting of: a        monomeric unit of the synthetic polymer hydrogel and another        P-X-R group, as defined herein, wherein the other P-X-R group        may be the same as or different from the first P-X-R group,        further wherein the dashed line is a covalent bond to the second        moiety if P is covalently attached to two moieties;    -   X is a covalent bond or a linking moiety; and    -   R is a functional moiety for attaching, either covalently or        non-covalently, a biomolecule.

The polymerizable attachment molecules, designated ‘P-X-R’, areincorporated into the permeation layer by copolymerization duringfabrication of the permeation layer, or fabrication of a portion of thepermeation layer, to produce a permeation layer containing copolymerizedattachment moieties.

The X bond or linking moiety is typically a covalent bond or standardspacer or linker group. If X is not a covalent bond, X is preferablyselected from the group consisting of an alkyl of 1-10 carbon atoms, analkenyl of 2-10 carbon atoms, alkyl esters, ketones, amides, thioesters,alkyl ethers, amido groups, carbonyls, and/or any combinations thereof.In some preferred embodiments of the invention, X comprises apolyethylene glycol moiety, while in other preferred embodiments of theinvention, X represents a covalent bond.

As used herein, alkyl denotes straight-chain and branched hydrocarbonmoieties such as methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl,sec-butyl, neopentyl, tert-pentyl and the like. Such alkyls may besubstituted with a variety of substituents including but not limited tohydroxy, oxo, amino, thio, cyano, nitro, sulfo and the like. Alkenyldenotes a hydrocarbon wherein one or more of the carbon-carbon bonds aredouble bonds and the non-double bonded carbons are alkyl or substitutedalkyl. Alkenyl hydrocarbons groups may be straight-chain or contain oneor more branches. Amino refers to moieties including a nitrogen atombonded to 2 hydrogen atoms, alkyl moieties and combination thereof.Amido refers to moieties including a carbon atom double bonded to anoxygen atom and single bonded to an amino moiety.

The R moieties on the attachment molecule are preferably a moietycapable of participating in a biotin-binding pair reaction, i.e., eithera biotin-moiety or a biotin-binding moiety. Biotin-binding moietiesinclude anti-biotin antibodies; versions of avidin or streptavidin whichhave been genetically engineered, enzymatically cleaved, or chemicallymodified; and other synthetic biotin-binding structures which bind tobiotin with a dissociation constant that is functionally equivalent toavidin or streptavidin. The R moieties in preferred embodiments of thepresent invention bind to derivatized biomolecules, which have beenderivatized to contain either a biotin-moiety or a biotin-binding moiety(e.g., streptavidin). As many biomolecules may be readily derivatizedwith biotin or a biotin analog, in preferred embodiments of theinvention R is a biotin-binding moiety such as, for example,streptavidin, avidin, or chemical or recombinantly engineeredderivatives thereof. As will be appreciated by those of skill in theart, the attachment molecule may contain more than one P moiety attachedto an R moiety, especially in the case of a proteinaceous R moietiessuch as streptavidin, which has a homotetrameric quaternary structure.In some preferred embodiments of the invention, the attachment moleculehas about one P moiety for each biotin-binding site on R, while in otherpreferred embodiments, the attachment molecule has less than one Pmoiety for each biotin binding site on R.

The R moiety is utilized to attach biomolecules or other bindingentities to the permeation layer at the microlocation. Severalattachment chemistries (both non-covalent affinity chemistries andcovalent reactive chemistries) have been developed for specificallyderivatizing biomolecules such as nucleic acids, proteins, andpolypeptides, an other molecules to allow for specific attachment toanother moiety while retaining the activity of the derivatized molecule.Generally, these include, covalently bonded chemical moieties fornon-covalent attachment such as streptavidin, biotin, phenyl boronicacid, salicylhydroxamic acid, or even synthetic binding entities such asspecific pyranosyl RNA sequences (or “pRNAs,” described in co-pendingapplication Ser. No. 09/374,338 filed Aug. 13, 1999, herein incorporatedfully by reference,) or reactive moieties for covalent attachment, suchas N-hydroxysuccinimidyl active esters, amines, aldehydes, acylchlorides, hydrazines, hydrazides, and the like. Derivatizations forattachment to R also include oligonucleotides containing oxidizedribose, amine terminations, or any of the well known bioconjugate pairsas outlined by Hermanson (Hermanson, G. T. Bioconjugate Techniquescopyright 1996, Academic Press, San Diego, Calif.) herein incorporatedby reference. Preferably, attachment of the chemical moieties forattachment to R to the biomolecule or other specific binding entitycomprises a covalent bond. However, attachment of the derivatizedbiomolecules to a copolymerized attachment moiety in the permeationlayer of the microarray may be through either a covalent or anoncovalent bond.

Thus, in alternative preferred embodiments, R is a reactive moiety forcovalent attachment of a derivatized biomolecule by amine, hydrazine, orhydrazide attachment chemistries, such as an active N-hydroxylsuccinimidyl (NHS) ester, a sulfonated NHS ester, an aldehyde, an acid,an acyl halide or the like, or a hydrazide, hydrazine, amine. Hydrazideattachment chemistries are detailed in PCT/US01/41663 (designating theUS), which are useful to attach binding entities to the mesoporoussynthetic hydrogel permeation layers of the devices of the invention. Inaddition, other reactive groups for use in common biomolecule attachmentchemistries may be used, such as those suitable for disulfide linkagesor thioester linkages (e.g., thiols), phosphorothiolate monoesters,acetals, ketones, aldehydes, dialdehydes, bromo- or iodo-acetamides, andesters. In addition, psoralens may be useful as R in various embodimentsof the invention. Preferred psoralens for use are photoactivatable at awavelength of about 365 nmn.

In other alternative embodiments of the invention, R may be a syntheticpairing system unit, such as a pyranosyl-RNA oligomer, for immobilizingthe specific binding entity at particular microlocations on the activeelectronic matrix device. In these embodiments, the pRNA may bederivatized with a polymerizable moiety and copolymerized into themesoporous synthetic polymer hydrogel permeation layer as is generallydescribed for P-X-R molecules. However, as described for copolymerizedspecific binding entities below, the use of a mold which producepermeation layers which cover just one microlocation electrode or asubset of microlocation electrodes, rather than an entire array ofmicrolocation electrodes, is preferred.

As described for biotin binding moieties, such as streptavidin, R isusually copolymerized in an active state. However, especially when usingreactive groups as R, R may be modified to require deprotection oractivation in order to function as an attachment moiety. For instance,t-butyl or another bulky moiety may be used to shield a reactive R groupduring polymerization, and then cleaved prior to the attachment of thespecific binding entities to the microlocations of the active electronicmatrix device. Alternatively, R can be provided as a reactive groupprecursor in the co-polymerized molecule, and then converted (e.g., byan oxidation or reduction reaction) to the reactive group.

The P moiety may react with monomers of the surrounding permeationlayer, or with P moieties of a second attachment molecule, and so forth.Thus, the attachment molecules to be added may also already be partiallypolymerized prior their incorporation into to the base permeation layer(e.g., in the form of a block copolymer, or other additive unit). Thepolymerization reaction may be carried out in a solution, slurry, orother acceptable format, where the P moiety may react with a monomermoiety of the growing permeation layer polymer (which may be the same as‘P’) and/or a P moiety of another attachment molecule. Thepolymerization of P into the permeation layer may be initiatednonspecifically by using a polymerization initiator molecule, such as afree radical polymerization initiator, that is sensitive to heat and/orspecific wavelengths of electromagnetic radiation. The use of suchactivatable initiators allows the initiation of polymerization by anoutside energy source (heat or a wavelength of light), which can beapplied across the entire microelectrode array, or at specific portionsof the microelectrode array.

P comprises a chemical moiety which includes a reactive center that mayparticipate in bonding to another P reactive center in a polymerizationreaction, and/or bond to a reactive center of the permeation layerpolymer matrix. Additionally, P may bond to an R moiety of anotherfunctional group. P is preferably selected from the group consisting ofalkenyl moieties including but not limited to substituted orunsubstituted α,β,unsaturated carbonyls wherein the double bond isdirectly attached to a carbon which is double bonded to an oxygen andsingle bonded to another oxygen, nitrogen, sulfur, halogen, or carbon;vinyl, wherein the double bond is singly bonded to an oxygen, nitrogen,halogen, phosphorus or sulfur; allyl, wherein the double bond is singlybonded to a carbon which is bonded to an oxygen, nitrogen, halogen,phosphorus or sulfur; homoallyl, wherein the double bond is singlybonded to a carbon which is singly bonded to another carbon which isthen singly bonded to an oxygen, nitrogen, halogen, phosphorus orsulfur; alkynyl moieties wherein a triple bond exists between two carbonatoms. More preferred P include substituted or unsubstitutedα,β,unsaturated carbonyls, vinyl, allyl and homoallyl groups andalkynes. P may also be selected from the group consisting of acetal,epoxide, ester, carboxylic acid, amide, halo-acetamide, thiol,phosphorothiolate monoester, thioester, disulfide, aldehyde, ketone,hydrazide, hydrazine, and amines. In more preferred embodiments, P is anacryloyl or acrylamido moiety. In especially preferred embodiments, P isan acrylamido moiety, and most preferably a methacrylamide moiety.

As will be appreciated by those of skill in the polymer arts, anappropriate P should be selected which will easily copolymerize withmonomers of the synthetic polymer hydrogel matrix, in order to ensureeven distribution and good incorporation of the attachment moietiesthroughout the synthetic polymer hydrogel permeation layer. Generally,the use of a P which is the same as or similar to the monomer of thepolymerization mixture will produce a better copolymerized attachmentmoiety containing mesoporous synthetic polymer hydrogel permeationlayer. Thus, in the examples below, an acryloyl or acrylamido moiety isused as P, for copolymerization into an acrylamide hydrogel.

Copolymerized Specific Binding Entities

In alternative preferred embodiments of the invention, the mesoporoussynthetic polymer hydrogel permeation layer comprises copolymerizedspecific binding entities. In these embodiments, the copolymerizedspecific binding entity has the same general formula:

as defined above, with the change that R is a specific binding entity.As discussed, specific binding entities useful for assays performed onthe active electronic matrix chip devices of the invention include, butare not limited to, nucleic acids, proteins, polypeptides,proteoglycans, glycoproteins, antigenic epitopes, and other moleculesuseful in biochemical or chemical assays. Preferred specific bindingentities include nucleic acids (e.g., DNA, RNA, chemically derivatizedDNA or RNA, or nucleic acid analogs which hybridize with naturallyoccurring nucleic acids), proteins, antibodies, and antigens. Asdemonstrated by the copolymerization of streptavidin in thepolyacrylamide mesoporous synthetic polymer hydrogel permeation layersof the examples, proteins and polypeptides may be readily derivatizedwith acryloyl or acrylamido groups (or other polymerizable moieties),and copolymerized with minimal loss of protein activity. In addition,several phosphoramidite reagents are available to facilely attach acopolymerizable moiety, such as an acryloyl or acrylamido group, toeither end of nucleic acids (e.g., Acrydite™ from Mosaic Technologies,Boston, Mass.). Thus, specific binding entities may be incorporated intothe permeation layer directly by copolymerization.

Because one usually does not wish to have the same specific bindingentity at each microlocation of an active electronic matrix device, itis advantageous to use molds for polymerizing the permeation layer ontothe active electronic matrix microelectrode array which are sized toproduce a permeation layer over one microlocation or a sub-set ofmicrolocations on the active electronic matrix device. Such molds mayeasily be designed for an array of electrodes as pictured in FIG. 1,wherein a plurality of mold cavities are provided in the mold whichalign with specific microelectrodes on the device. For instance, suchmolds may comprise an array of mold cavities of an appropriate shapewhich at least cover the individual microlocation electrodes of theactive electronic matrix chip device (for instance, 100 round, square,or hexagonal cavities which are at least 80 μm in diameter for theelectrodes in FIG. 1). Or, molds may be produced which comprise cavitieswhich cover a sub-set of microlocation electrodes (e.g., an entire row,or a square of 4 electrodes, etc.). The polymerization mixturecomprising a particular copolymerizable specific binding entity may thenbe pipetted into those mold cavities which correspond to themicroelectrodes of the array which are to be covered with a mesoporoussynthetic polymer hydrogel permeation layer comprising the particularspecific binding entity. Such molds may be fabricated in quartz, glass,or other materials using standard microlithographic techniques, or maybe molding in plastics from a master created using such techniques.

The use of copolymerized specific binding entities is particularlyattractive for the production of clinical application active electronicmatrix chip devices, in which several pre-loaded specific bindingentities may be provided to test for a panel of particular nucleic acidsequences, antibodies, or antigens in a sample. As clinical users havean interest in convenience and time-savings for test devices, suchpre-loaded active electronic matrix chip devices offer significantadvantages over generic devices which must be loaded with specificbinding entities by the consumer. In addition, the copolymerizedspecific binding entity devices of the invention are also useful inresearch contexts in which several samples will be assayed for the samenucleic acid sequences, antibodies, or antigens in a several samples forcomparison. By copolymerizing the specific binding entity into thepermeation layer over a set of microelectrodes, the specific bindingentity concentration is standardized for that set of microlocations,allowing for more rigorous comparison of the binding of the analytebetween samples. Because the micromolding process lends itself readilyto automated steps in which polymerization mixtures containing theparticular copolymerizable specific binding entities (e.g., particularnucleic acid probes covalently linked to an acryloyl moiety) can beinterchanged, the manufacture of custom pre-loaded chips for particularresearch experiments may be easily realized.

Basic Active Electronic Matrix Chip Design

In order for an active electronic matrix chip device to carry outmulti-step and multiplex reactions, its electronic components must beable to maintain active operation in aqueous solutions. To satisfy thisrequirement, each microlocation has an underlying controllable andfunctioning DC micro-electrode. However, it is important for deviceperformance, particularly sensitivity (signal to noise ratio), thatbinding and affinity reactions are not prevented by the electrolysisreactions occurring on the active DC electrode surfaces. In addition tothe damaging effects incurred by any of the sensitive reagents andanalytes (DNA, RNA, proteins, etc.) directly contacting the electrodesurface, the electrodes produce electrolysis products which include acid(H⁺), base (OH⁻), hydrogen, oxygen, and various free radical specieswhich can also damage the sensitive components. Other considerations forthe design and fabrication of a device include, but are not limited to,materials compatibilities (including compatibility with the permeationlayer and its manufacture, and solution components utilized in variouschemical or biochemical assays on the device), nature of the specificbinding entities and the subsequent reactants and analytes, and thenumber of microlocations.

By “a controllable and functioning DC mode micro-electrode” is meant amicro-electrode biased either positively or negatively, operating in adirect current mode (either continuous or pulse or DC/AC), which can ina controllable manner affect or cause the free field electrophoretictransport of charged specific binding entities, reactants, or analytesto or from any location on the device, or from the sample solution.

As described herein, the free field electrophoretic “transport” ofmolecules is not dependent on bounding or confining the electric fieldproduced by an insulating material. Conventional electrophoreticseparation technologies require confinement or enclosure of electricfield lines by insulating (non-conducting) materials (e.g., the sides ofa glass capillary tube in capillary gel electrophoresis). In the case offree field electrophoretic transport on active electronic matrix chipdevices, charged molecules are moved from one microlocation through thebulk solution volume to any other microlocation, or from the bulksolution to specific microlocations. Therefore, special arrangements orconfinement by insulating materials is not required for this aspect ofthe invention. However, the relatively small area of the microlocationtest site allows high current densities to be produced. These highcurrent densities over a confined area of the chip allow for the rapidconcentration of the charged nucleic acids or other biomolecules fromsolution and electronic stringency for de-hybridization.

An active electronic matrix chip device can be designed to have as fewas two addressable microlocations or as many as hundreds of thousands ofmicrolocations. In general, a complex device with a large number ofmicrolocations is fabricated using microlithography techniques orcombination of microfabrication and micromachining. Fabrication iscarnied out on silicon or other suitable substrate materials, such asglass, silicon dioxide, plastic, insulated metallic or ceramicmaterials. These microelectronic “chip” designs would be consideredlarge scale array or multiplex analysis devices.

FIG. 1 shows the electrode array of a 100 site NanoChip® device,produced using photolithographic techniques. The microlocations are theareas in and on the permeation layer above the exposed metal electrodepads, which have been deposited on an insulator layer/base material. Themetal pads serve as the underlying micro-electrode structures. Electrodematerials can include but are not limited to: aluminum, copper, carbon,iron, silver, gold, palladium, platinum, titanium, tungsten,polysilicon, and indium tin oxide, as well as silicide materials such asplatinum silicide, titanium silicide, gold silicide, or tungstensilicide. Special techniques known in the art for ensuring properadhesion to the insulating substrate materials (SiO₂) are used withdifferent metals. Various metals and other materials may be used fordifferent conductive components of the device, for example, usingaluminum for the perimeter contact pads, titanium for the interconnectcircuitry, and a noble metal (gold or platinum) for themicro-electrodes. In addition, it may be advantageous to use a layeredelectrode structure, in which a surface layer of a less reactive noblemetal, or noble metal alloy is layered over another metal layer. Forinstance, in the electrodes shown in FIG. 1, the electrode is platinumdeposited onto a support layer of titanium/tungsten alloy (for improvedadhesion). Alternatively, conductive polymers such as polyanilines orpolypyrrolidines may be used as electrode materials. In addition to theelectrodes underlying the microlocations of the active electronic matrixchip devices, other electrodes may be provided which do not underliemicrolocations on the chip devices. These include, for example, counterelectrodes which are simply used as a counter-biased electrode duringthe electrophoretic transport of the biomolecules in the solution. Asthey are not utilized as microlocation electrodes, they are usually notcovered by the permeation layer. An example of these electrodes is shownin FIG. 2, in which the counter electrodes are positioned in a circlesurrounding the center array of microelectrode pads underlying themicrolocations of the chip device. In addition to the counterelectrodes, other electrodes, such as reference or pseudo-referenceelectrodes (e.g., silver paste electrodes, etc.) for use in measuringand maintaining constant current or voltage supplied to the electrodes,may be incorporated into the chip device.

On the surface of the finished chip device, an insulator materialseparates the metal electrode pads from each other in the plane of thechip device. This separation of the electrodes by the insulatingmaterial prevents the “short circuiting” of the electron current throughthe surface of the chip device, rather than being directed through thesolution over the chip device. Generally, the insulator material (e.g.,silicon dioxide) is a layer deposited over and between the metal layerdeposited on the substrate, or a sandwich-type electrode and lead wirestructure, as shown in FIG. 1. In this manufacturing strategy, theinsulating layer deposited above the electrode pads is removed to exposethe functioning micro-electrodes. This can be more easily seen in FIG.2, where the exposed electrode sections appear as darker circles abovethe conductive electrode material. The darkened appearance is caused byincreased depth of the permeation layer in the etched “holes” in theinsulating silicon dioxide layer. However, other manufacturingstrategies will be apparent to those in the microelectronic arts.Insulator materials include, but are not limited to, silicon dioxide,silicon nitride, glass, resist, polyimide, rubber, plastic, or ceramicmaterials. The microelectrodes on the NanoChip® device shown areseparated by a silicon dioxide layer, which also covers and insulatesthe micro wiring which connects the electrode pads to electricalconnections on the perimeter of the chip (outside of FIG. 1).

Thus, the basic features of an individual microlocation formed bymicrolithographic techniques are: the metal pad, which defines thelocation of the addressable microlocation, and which may incorporate alayer for the covalent attachment of the permeation layer; a permeationlayer overlaying the microelectrode; an attachment layer, which may becoextensive with the permeation layer, or another layer ofattachment-moiety laden permeation layer material overlaying a basepermeation layer.

The thickness of the permeation layer for microlithographically produceddevices can range from approximately 1 nanometers (nm) to 100 microns(μm), with 2 nm to 10 μm being the most preferred. Generally, inpreferred embodiments of the permeation layers of the invention, thepermeation layers are between about 0.5 μm and about 10 μm thick in thedry state, more preferably between about 1.0 μm and about 5.0 μm thickin the dry state, and most preferably between about 1.0 μm and about 2.0μm thick in the dry state (or at least about 8-9 μm thick when inequilibrium with aqueous buffer). Also, in preferred embodiments, thepermeation layers vary in thickness across the electrode array of theactive electronic matrix chip device by less than 0.5 μm, morepreferably by less than 0.2 μm, and most preferably by less than 0.1 μm,as measured in the dry state.

As mentioned above, synthetic polymer hydrogel permeation layers may bemolded onto the active electronic matrix microelectrode array as asingle layer, or as a plurality of layers. In some cases, the permeationand attachment layers can be formed from the same material, asdemonstrated in the Examples. Alternatively, a base permeation layer maybe molded onto the microelectrode array, followed by a permeation layercontaining copolymerized attachment moieties or copolymerized specificbinding entities. When more than one layer forms the permeation layer,it is preferred that subsequent layers be covalently attached to (orgrafted onto) the previous layer. The use of multi-layer permeationlayers is especially preferred when using higher cost or specialtyreagents, such as copolymerizable specific binding entities.

Optimally, the attachment layer has from 10⁵ to 10⁷ attachment moieties(or specific binding entities, after attachment) per square micron (μm²)for the attachment of specific binding entities. Thus, whencopolymerized attachment moieties are provided in the mesoporoussynthetic polymer hydrogel permeation layer devices of the invention,they should be present at sufficient concentration in the polymerizationmixture to provide a density of attachment sites within this range. Theattachment of specific binding entities should not overcoat or insulatethe surface so as to prevent the underlying micro-electrode fromfunctioning. A functional device requires some fraction (˜5% to 25%) ofthe actual metal micro-electrode surface to remain accessible to solvent(H₂O) molecules through the permeation layer, and to allow the diffusionof counter-ions (e.g., Na⁺ and Cl⁻) and electrolysis gases (e.g., O₂ andH₂) to occur. The intermediate permeation layer is also designed toallow diffusion to occur. Additionally, the permeation layer should havea pore limit property which inhibits or impedes the larger bindingentities, reactants, and analytes from physical contact with themicro-electrode surface. Thus, the permeation layer keeps the activemicro-electrode surface physically distinct from the binding entitylayer of the microlocation.

Addressable microlocations can be of any shape (e.g., such as round,square, or rectangular.) The size of an addressable microlocation can beof any size, preferably range from sub-micron (˜0.5 μm) to severalcentimeters (cm), with 5 μm to 100 μm being the most preferred sizerange for devices fabricated using microlithographic techniques. Themicrolocation size for the device shown in FIG. 1 is 80 μm. The spacingbetween microlocations is determined by the ease of fabrication, therequirement for detector resolution between microlocations, and thenumber of microlocations desired on a device. For instance, the distancebetween the microlocations of the chip device shown in FIG. 2 is 120 μmedge to edge, or 200 μm center to center. However, particular spacingsbetween microlocations, or spatial arrangement or geometry of themicrolocations, are not necessary for device function, in that anycombination of microlocations (i.e., underlying micro-electrodes) canoperate over the complete device area. As the number of microlocationsincreases beyond several hundred, the complexity of the underlyingcircuitry of the microlocations increases. In this case themicrolocation grouping patterns have to be changed and spacing distancesincreased proportionally, or multi-layer circuitry can be fabricatedinto the basic device, i.e., transistors and semiconductor controlelements incorporated directly into the silicon. As mentioned above,connective circuitry for each individual underlying micro-electrodepreferably runs to an outside perimeter of metal contact pads. Whenthese contact pads are produced in a standard configuration, the chipdevice can be mounted in a standard quad package, and the chip contactpads wired to the quad package pins.

It is not necessary to enclose the device or completely confine themicrolocations with dielectric or insulating barriers because complexelectronic field patterns or dielectric boundaries are not required toselectively move, separate, hold, or orient specific molecules, in thespace or medium between any of the electrodes. The active electronicmatrix accomplishes spatial separation by attaching the specific bindingmolecules and subsequent analytes and reactants to the surface of anaddressable microlocation. However, it is preferred that the activeelectronic matrix chip devices of the invention be contained, orpackaged, within a flow-cell to allow for the controlled introduction ofsamples and reagents onto the surface of the devices. In general, it ispreferred that such flow cells comprise a closed volume over the activeelectronic matrix chip device with at least one input port and at leastone output port. In this manner, the functions of sample introduction,rinsing, and reagent introduction may be easily carried out on theactive electronic matrix chip devices using standard fluidicstechniques.

Systems containing more than one chip and additional packaging andperipheral components may be designed to address problems related toclinical diagnostics (i.e., addition of sample materials, fluidtransfer, and containment of bio-hazardous materials, and the detectionof labeling moieties such as, for example, fluorescent,chemiluminescent, colorigenic, or radioactive moieties.) The packagedchip can then be plugged into a microprocessor controlled DC powersupply and multimeter apparatus which can control and operate thedevice. It is contemplated by this invention that device manufacture(prior to addressing) may involve the incorporation of various basiccomponents into a disposable device which would be essentiallysandwiched together in an assembly such as: the basic chip device towhich the binding entities are attached; a flow cell fluid containmentcomponent; and, optionally, on board active electronic matrix controllercomponent. This strategy solves a number of problems related tofabrication techniques and materials compatibilities.

Anchoring Chemistries for the Permeation Layer

Although synthetic polymer hydrogel materials such as those noted abovehave desired functional qualities, hydrogel permeation layers are proneto separate or ‘delaminate’ from the electrode surface. Although theapplicants are not bound by any particular theory, this delamination isthought to be caused by a change in the chemical make-up at theinterface between the permeation layer and the electrode resulting fromthe application of electronic potential at the electrode and by physicaldisruption from charged ions and gases emanating from the electrode.Such delamination can be viewed from the standpoint of‘microdelamination’ and ‘macrodelamination’.

Microdelamination involves the electrochemical degradation of thechemical interface between the permeation layer and the electrodeitself. It is observed by the formation of raised bulges in thepermeation layer, or by ringlets visible due to defraction of light fromthe delaminated layer when appropriately viewed by a confocal microscopeand results in the loss of consistency in permeation layer performance(possibly due to the loss of control over the electric fielduniformity). Macrodelamination, on the other hand, is caused by amismatch of the surface energies between the permeation layer and thechip substrate and results in permeation layer peeling (lift-off) whichcan extend across the entire microchip surface. Since the permeationlayer provides a means for the attachment of specific binding entitiesfor analytes present in the liquid overlay, macrodelamination isdetrimental to assays typically run on the active electronic matrix chipdevices. In preferred embodiments of the synthetic polymer hydrogelactive electronic matrix chip devices of the invention, the problem ofmicro- and macrodelamination may be alleviated by the use of a covalentchemistry linkage between the active electronic matrix electrode arrayand the permeation layer hydrogel matrix. This chemistry is applicableto a variety of permeation layer synthetic polymer hydrogelcompositions, including polyacrylamides, and is able to withstandcurrent densities of at least 0.04 nA/μm² and/or voltage drops between 1and 3 V.

In the case of metals like aluminum or silicon/noble metal mixtures, themetal oxide layer provides a base for the covalent coupling of thepermeation layer. Metal oxide and hydroxyl groups (either alone or incombination), and other similar interface surfaces known to thoseskilled in the art of surface coating chemistries provide covalent sitesfrom which to construct or hold the permeation layer. Preferredmetal/silicide electrodes include platinum silicide (PtSi), tungstensilicide (WSi), titanium silicide (TiSi), and gold silicide (AuSi), asthey provide sites which may be readily utilized for silane couplingusing the reagents noted below.

However, it is not essential that the permeation layer be covalentlyanchored to the metal electrode surface. Significant additionalresistance to micro- and macro-delamination may be obtained by simplyattaching the permeation layer to the substrate or insulating materialsurrounding the actual microelectrode of the microlocation. The physicaloverlaying of permeable materials represents an alternative method whichis also within the scope of this invention. Thus, in some preferredembodiments, the synthetic polymer hydrogel permeation layers of thedevices of the invention are covalently anchored to the substrate of theactive electronic matrix device which surrounds the microelectrode. Asused to describe this method, the “substrate” is considered to includenot only a layer of support material that the microelectrode is formedon, but also any insulating material layers (e.g., silicon dioxide)which may overly the metal of the electrode and thus define the boundaryof the microlocation. For example, in the case of metals like platinumand gold, a permeation layer can be physically overlaid, and thenanchored to the silicon dioxide layer surrounding the electrode. This“tenting” approach, described in Example 1, has proven very useful inpreventing macrodelamination of the permeation layer. The use ofcross-linking agents to anchor a permeation layer to an electrode of anactive electronic matrix chip device is generally described in U.S. Pat.No. 6,303,082, incorporated fully herein by reference.

In an example of these preferred embodiments, the covalent attachmentcomprises a linking moiety that provides an moiety for bonding thelinker to the silanol moiety of a silicon bearing surface (e.g., ametal/Si electrode or a silicon containing surface of the substrate) anda separate moiety for bonding the linker to the permeation layer. In aparticularly preferred embodiment, the linking moiety is defined by theformula:

where X=acrylate, methacrylate, acrylamide, methacrylamide, allyl,vinyl, acetyl, amine (substituted or not), epoxy or thiol;

SPACER=alkyl, aryl, mono- or polyalkoxy (such as ethyleneglycol orpolyethyleneglycol), mono- or polyalkylamine, mono- or polyamide,thioether derivatives, or mono- or polydisulfides;

A and B=any combination of Oxygen-R, where R=H, alkyl such as methyl,ethyl, propyl, isopropyl or other linear or branched hydrocarbon, Cl, Bror a moiety functionality similar to that of X-SPACER; and

C=Oxygen-R, where R=H, alkyl such as methyl, ethyl, propyl, isopropyl orother linear or branched hydrocarbon, Cl, Br, or any other hydrolyzablemoiety.

In embodiments where metal/Si microlocation electrodes are used, thesilanol moiety can react with hydroxyl groups bonded to a silicon moietyof the electrode surface. In embodiments where a silicon-containinggroup is not present on the electrode, but is present on the substrate,the silanol moiety can react with hydroxyl groups bonded to a siliconmoiety on the substrate surface. On the other end of the linker, the Xmoiety comprises chemical groups that are available to covalently reactwith reactive centers of the perneation layer polymer.

As shown in FIG. 4, the permeation layer may be linked to the electrodeby a linking moiety that has at least one copolymerizable reactivecenter. Linkers having suitable characteristics are provided in theTable below:

CHEMICAL TYPE FORMULA ACRYLATES: CH₂═CHCOOCH₂CH₂CH₂Si(OCH₃)₃CH₂═CHCOOCH₂CH₂CH₂SiCl₃ CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)(OCH₃)₂CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)₂(OCH₃) CH₂═CHCOOCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═CHCOOCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃ METHACRYLATES:CH₂═C(CH₃)COOCH₂CH₂CH₂Si(OCH₃)₃ (MOTS) CH₂═C(CH₃)COOCH₂CH₂CH₂SiCl₃CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)(OCH₃)₂CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)₂(OCH₃) CH₂═C(CH₃)COOCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═C(CH₃)COOCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃ ACRYLAMIDES:CH₂═CHCONHCH₂CH₂CH₂Si(OC₂H₅)₃ (AMPTS) CH₂═CHCONHCH₂CH₂CH₂SiCl₃CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)(OCH₃)₂ CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)₂(OCH₃)CH₂═CHCONHCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═CHCONHCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃CH₂═CHCONHCH₂CH₂CONHCH₂CH₂CONHCH₂CH₂CH₂Si(OC₂H₅)₃ METHACRYLAMIDES:CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(OCH₃)₃ CH₂═C(CH₃)CONHCH₂CH₂CH₂SiCl₃CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)(OCH₃)₂CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)₂(OCH₃) CH₂═C(CH₃)CONHCH₂CH₂CH₂Si(CH₃)Cl₂CH₂═C(CH₃)CONHCH₂CH(OH)CH₂NHCH₂CH₂CH₂Si(OC₂H₅)₃ ALLYL DERIVATIVES:CH₂═CHCH₂NHCH₂CH₂CH₂Si(OCH₃)₃ CH₂═CHCH₂SiH(OCH₃)₂ CH₂═CHCH₂Si(CH₃)₂ClCH₂═CHCH₂SiHCl₂ CH₂═CHCH₂Si(OCH₃)₃ AMINO DERIVATIVES:H₂NCH₂CH₂NHCH₂CH₂CH₂Si(OCH₃)₃ (AEAPS)H₂NCH₂CH₂CH₂CH₂CH₂CH₂NHCH₂CH₂CH₂Si(OCH₃)₃ (AHAPS) H₂NCH₂CH₂CH₂Si(OCH₃)₃(APS) H₂NCH₂CH₂CH₂Si(OC₂H₅)₃ EPOXY DERIVATIVES:

In a particularly preferred embodiment, active electronic matrix chipshaving covalent attachment chemistry use a linker selected from thegroup consisting of: APS, AEAPS, AHAPS, MOTS, and AMPTS.

Example 1 illustrates the use of a silanol linker to anchor thepermeation layer to the substrate surrounding a platinum microlocationelectrode. Alternatively, if a noble metal/silicide electrode is used,the electrode array of the chip may be first treated with an argonplasma for 5 minutes at 250 mTorr and 250 Watts. The chip may then betreated with the linker by vapor deposition over 15 minutes at roomtemperature then cured onto the chip by heating for 2 hours at 90° C.This causes the linker to covalently bind to the hydroxyl groups of thesilicide moiety in the electrode. Once the linker is attached to themicrochip, a UV-initiated free radical polymerization reaction can beconducted between the monomers which will make up the permeation layerand the vinyl moieties present at the surface of linker-derivatizedelectrodes, thereby synthesizing the permeation layer and covalentlyanchoring it to the electrode in a single step.

Applications Utilizing Active Electronic Matrix Devices With MesoporousSynthetic Hydrogel Permeation Layers

In general, the mesoporous synthetic hydrogel permeation layer devicesof the present invention may be addressed and used in much the samemanner as previously disclosed active electronic matrix chip devices. Adescription of the electronic addressing procedure may be found in U.S.Pat. No. 6,051,380. Briefly, the devices may be addressed by introducinga solution containing an attachment-derivatized molecular species to beaddressed onto the active electronic matrix chip device (usually in aflow-cell compartment), and biasing the electrodes under themicrolocations to which the molecular species is to be addressed, sothat the electrode is biased opposite the charge of the molecularspecies (e.g., positive if the molecular species is a nucleic acid). Themolecular species then migrates through the solution to themicrolocation, and becomes attached via the attachment-facilitatingmoiety to an attachment moiety in the permeation layer (e.g., through abiotin-streptavidin interaction). As described in U.S. Pat. No.5,051,380, preferred solutions for use are low-conductance buffers withsignificant buffering capacity, such as histidine (preferably about 50mM) or other zwitterionic buffers. In this manner, nucleic acid probes,sample nucleic acids, amplicons from samples, antigens, antibodies, orany other charged molecular species, may be addressed to specificmicrolocations of the device, and attached for use in various assayformats.

After addressing, the mesoporous synthetic hydrogel permeation layerdevices may be utilized in a multitude of assay formats. For instance,devices which are addressed with nucleic acid probes or amplicons may beutilized in dot blot or reverse dot blot analyses as described in U.S.Pat. No. 5,051,380, base-stacking single nucleotide polymorphism (SNP)analysis as described in U.S. Ser. No. 09/291,129, SNP analysis withelectronic stringency as described in U.S. Ser. No. 09/727,030, or inSTR analysis as described in U.S. Pat. No. 6,207,373. In addition, suchaddressed devices may be utilized in formats for enzymatic nucleic acidmodification, or protein-nucleic acid interaction, such as, e.g., geneexpression analysis with enzymatic reporting as described in U.S. Ser.No. 09/710,200, anchored nucleic acid amplification as described in U.S.Pat. No. 6,238,868, or other nucleic acid modifications suitable forsolid-phase formats including restriction endonuclease cleavage, endo-or exo-nuclease cleavage, minor groove binding protein assays, terminaltransferase reactions, polynucleotide kinase or phosphatase reactions,ligase reactions, topoisomerase reactions, and other nucleic acidbinding or modifying protein reactions.

In addition, the mesoporous synthetic hydrogel permeation layer devicesare generally useful in immunoassay formats. For instance, themicrolocations of the devices can be addressed with antigens (e.g.,peptides, proteins, carbohydrates, lipids, proteoglycans, glycoproteins,etc.) in order to assay for antibodies in a bodily fluid sample bysandwich assay, competitive assay, or other formats. Alternatively, themicrolocations of the device may be addressed with antibodies, in orderto detect antigens in a sample by sandwich assay, competitive assay, orother assay formats. As the isoelectric point of antibodies and proteinscan be determined fairly easily by experimentation or pH/chargecomputations, the electronic addressing and electronic concentrationadvantages of the devices may be utilized by simply adjusting the pH ofthe buffer so that the addressed or analyte species will be charged.

In addition to the simple active electronic matrix assay formatsdescribed above, formats utilizing specific pairing components asderivatization moieties for the attachment of biomolecules have alsobeen described for active electronic matrix chip devices. These formatsallow the simultaneous electronic addressing of several molecularspecies to a set of microlocations by using the specific pairingcomponent (e.g., a pyranosyl RNA oligomer) to specifically bind to acomplementary specific pairing component which has been previouslyattached to the permeation layer at the microlocation. These formats candramatically reduce the amount of time necessary to create arrays ofdozens of distinct binding entity compositions on an active electronicmatrix array. The mesoporous synthetic hydrogel permeation layer devicesof the invention would also be useful in these formats, which have beendescribed for immunoassay-type arrays (see U.S. Ser. No. 09/783,763),and nucleic acid arrays (see U.S. Ser. No. 09/910,469).

All patents, patent applications, and published patent applications, andother publications referred to herein are hereby incorporated herein intheir entirety by reference, as if they were fully reproduced herein.

EXAMPLES

The invention will now be described in greater detail by reference tothe following non-limiting examples regarding the production and use ofmesoporous synthetic polymer hydrogel permeation layers on activeelectronic matrix devices.

The recipes for buffers, solutions, and media in the following examplesare described in J. Sambrook, E. F. Fritsch, and T. Maniatis, MolecularCloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

Example 1 Exemplary Mesoporous Synthetic Hydrogel Permeation Layers

A. Acryloyl or Acrylamido Streptavidin Production

For incorporation into the mesoporous synthetic hydrogel permeationlayer formulations, functionalized streptavidin derivatives with pendantacryl groups for incorporation into the polymer backbone were prepared.Streptavidin was derivatized with either an acrylamido group, or anamide-linked PEG 3400-ester-acryloyl group, according to the reactionscheme in FIG. 3. A N-acryloxysuccinimide was from Polysciences, Inc.and was used as received. α-Acryloyl, ω-N-hydroxysuccinimidyl ester ofpoly(ethylene glycol)—propionic acid, (M.W. 3400) (PEG-NHS) was fromShearwater Polymers, Inc. Streptavidin (SA) was purchased from RocheMolecular Biochemicals.

Native SA (165 mg) and an excess of NHS reagent (2.4 mg) were weighedout into a 15-ml disposable polypropylene cell culture tube. Sodiumphosphate buffer (50 mM, pH 9, 12 ml) was added and the tube wasvortexed briefly until all the solids were fully dissolved. The reactionwas allowed to continue for 30 minutes in the dark. Products ofstreptavidin derivatization reactions were purified using a gelpermeation column (HiPrep 26/10 GPC desalting column, Amersham PharmaciaBiotech) on an ÄKTA™ explorer HPLC system (Amersham Pharmacia Biotech).Elution was performed with sodium phosphate (50 mM, pH 7) at a rate of10 ml/min. The first peak eluted between 10 and 24 ml and the secondpeak eluted above 30 ml. The second peak contained the free NHS and theexcess acrylic acid that resulted from the SA derivatization reactionand from hydrolysis. All the fractions under the first peak werecollected and concentrated on a rotaro-evaporator (SpeedVac) at 35° C.for six to seven hours. The concentrated solution was brought to roomtemperature and the protein concentration was measured by UVspectrophotometery at 280 nm. The concentration was adjusted to 35 mg/mlwith deionized water.

The relative binding activities of unmodified SA, the N-acryl SA and thePEG-acryloyl SA were determined by a standard HABA assay. Unmodified SAhad a HABA activity of 4.0, while the N-acryl SA had an activity of 3.8.The PEG-acryloyl SA had a relatively low activity of 3.0, possiblybecause of steric hindrance by the bulky PEG group. Because of its highretained binding activity, the N-acryl SA was utilized in the testformulations in a multi-batch production round as described in Example3, and characterized in FIG. 10.

B. Activation of the Active Electronic Matrix Surface for CovalentPermeation Layer Anchoring

In order to improve adhesion of the permeation layer to the activeelectronic matrix chip surface, the surface was silanized with anacryloyl moiety (Bind-Silane™ (3-methacryloxy-propyl-trimethoxysilane),product of Pharmacia Biotech). The active electronic matrix substrateswere cleaned by argon plasma etching (100 W, for 30 min) andsubsequently reacted with Bind Silane™ under vapor phase conditions. Thevapor phase silanization was carried out in a polystyrene petri dish(150×15 mm). Five substrates were placed centro-symmetrically in thedish. Water (200 μl) was dispensed into the dish at 5 differentperimeter locations. Bind Silane™ (200 μl) was introduced into a smallpetri dish (35×10 mm) placed at the center of the large dish. The largepetri dish was closed and the reaction was allowed to continue for 15min at room temperature. The substrates were taken out and placed inglass petri dishes for subsequent heating in an oven at 90° C. for twohours.

One way of improving the effective function of a hydrogel as apermeation layer on an active electronic matrix chip is to have a strongadhesion between the hydrogel and the chip. By pre-treating the chipwith silane having polymerizable groups which can subsequently be linkedto a hydrogel via copolymerization, covalent linkage of the hydrogel tothe substrate surface can be achieved. Bind Silane™(3-methacryloxy-propyl-trimethoxysilane) is readily adsorbed onto theplasma cleaned active electronic matrix. The adsorbed silanesubsequently reacts with the surface hydroxyl groups produced by theplasma etching on the area around the micro electrodes on the chip,leading to the formation of a stable silanized surface. In order toensure complete surface attachment, the substrates after silaneadsorption were heated at 90° C. for two hours in an air oven. Thisprocess results in the deposition and immobilization of an oligolayer(10-100 layers) of the silane. Similar studies on the vapor phasesilanization of surfaces with (3-Aminopropyl)triethoxysilane (APS) haveshown that adsorption of neat APS vapors results in a stable chemisorbedlayer as well as a less stable physisorbed phase which can be removed byevacuation.

C. Exemplary Polymerization Mixture Formulations

Applicants developed several polyacrylamide-based permeation layerformulations for use on the active electronic matrix chips, arepresentative set of which are shown here. Although a single basepolymer formula is used, alternative formulas could also be devised bythose of skill in the art which would function well as the syntheticpolymer component of the permeation layer. Similarly, althoughderivatized SA is copolymerized with the permeation layers described inthese examples, other appropriate attachment moieties could becopolymerized with the permeation layer if non-biotin attachment [e.g.,covalent attachment chemistries or salicyl hydroxamic acid/phenylboronic acid chemistries] is to be utilized to address themicrolocations. Likewise, the attachment moieties could be completelyomitted if the permeation layer is to be later surface-derivatized, oran attachment layer is to be overlaid on a base permeation layer. Forthe reasons described above, however, it has been found to be preferableto copolymerized the attachment moieties into the permeation layer in asingle layer.

The production of the acryl derivatives of SA is described above.Polyoxyethylene 100 stearyl ether (Brij 700) was purchased from Sigma.DMSO was an Aldrich product. Darocur® 4265 was supplied by Ciba. Themonomer solutions were prepared by simply mixing the components togetherand vortexing, with the exception that Darocur was first dissolved inDMSO before addition to the other components. The basic formula used forthe exemplary permeation layers described herein was an aqueous solutionof:

total acrylamide/methylene bisacrylamide 18% w/v, 10 mol % bisacrylamide

acryloyl-PEG-SA 14 mg/ml

Darocur (added as 1.9% w/v solution in DMSO) 0.2% w/v

Brij 700:

-   -   Formula 4012-1: 0 mg/ml    -   Formula 4012-2: 11 mg/ml    -   Formula 4012-3: 18 mg/ml    -   Formula 4006-1: 73 mg/ml        Formula 4012-1 serves as a control synthetic polymer hydrogel        composition in the following functional experiments, while        4012-2, 4012-3, and 4006-1 demonstrate the effect of increasing        amounts of templating porogen.        D. Micromolding of the Synthetic Polymer Hydrogel Permeation        Layers onto the Active Electronic Matrix Chip Surface

A specially designed molding system was developed for the precisecontrol of both the hydrogel layer thickness and the uniformdistribution of UV radiation intensity. The system is comprised of aquartz microreaction mold with a precision-formed depression produced bystandard microlithography techniques, and a UV light source assembly. Asolution of the polymerization mixture (0.35 μl) was dispensed onto themold surface and the microelectrode array substrate was compressedagainst the mold at a pressure of approximately 1N. The polymerizationwas carried out in two steps, at an initial intensity of 700 μW/cm² for15 sec followed by at a higher intensity of 70 mW/cm² for an additional15 sec. The chip was withdrawn from the mold surface, washed in a streamof distilled water for 5 seconds and dried under a stream of nitrogen.This process was then repeated for the next chip.

The in situ polymerization and deposition of the hydrogel on the chipsurface is illustrated by the reaction scheme in FIG. 4. The reactionmolding is a controlled process which provides excellent thicknesscontrol of the molded layers. In addition, the system is designed tocontrol the UV intensity, further allowing control of the polymerizationrate and the degree of phase separation of the hydrogels. A stereomicroscopic dark field image of a molded hydrogel on a 10×10 arraysubstrate is shown in FIG. 2. The thickness control over a batch of 136molded permeation layers was tested. The average thickness was quiteuniform, being 1.8±0.3 μm. The polymerization conditions (UV intensityand exposure time) were optimized using a photo DSC. An optimum phaseseparation between the forming polymer and the surfactant templateduring polymerization has been achieved by using a two step UV curingprocess: an initial exposure to a low intensity (700 μW/cm² for 15seconds), followed by a high intensity exposure (70 mW/cm² for 15seconds.) Initiating the polymerization at a lower light intensity wasalso found to minimize the formation of gas bubbles from the exothermicpolymerization reaction.

As one of ordinary skill in the art will appreciate, this process lendsitself easily to automation, with robotics designed for the dispendingof the monomer solution into the mold, positioning the chip over themold, pressing the chip against the mold, irradiating the mold, and soforth. Thus, this type of process may be easily adapted tohigh-throughput production of the active electronic matrix chip devices.

In order to achieve complete removal of the surfactant, the substrateswere washed extensively with water. They were placed in distilled waterin a petri dish with shaking overnight, followed by rinsing thoroughlywith running distilled water. This proved sufficient to remove the Brijsurfactant material, leaving behind the mesostructured synthetic polymerhydrogel permeation layer.

Example 2 Scanning Electron Microscopy of the Mesoporous SyntheticPolymer Hydrogel Permeation Layer Structure

The morphology of the mesoporous synthetic polymer hydrogel permeationlayers was examined using a scanning electron microscope (SEM). Samplesfor SEM were prepared by gradual exchange of the water for ethanol in agradient mode and subsequent drying by critical point drying techniquewith supercritical CO₂. This procedure has been reported to maintain thegel structure of the polymer with very little alteration. The scanningelectron micrographs are shown in FIGS. 5A & B (4012-1), 6A & B(40012-2), 7A & B (4012-3), and 8A & B (4006-1). These micrographsclearly show the microstructure of the gels in the 5,000× scan, and showthe mesostructure of the gels in the 45,000× scan. From these pictures,the physically heterogeneous nature of the mesoporous synthetic polymerhydrogel permeation layers is evident, especially when compared to therelatively physically homogeneous nature of 4012-1.

When examined under 5,000× magnification, 4012-1 (no pore template)appeared to have a relatively smooth, nonporous morphology when comparedto the other permeation layers. 4012-3 and 4006-1 exhibited a veryporous structure with a wide distribution of larger pores. The pores inthe gel structures can be roughly divided into three classes:

-   -   Class I—pores of size less than 100 nm (nanoporous)    -   Class II—pores in the order of 100-1000 nm, mainly in the range        of 100-500 nm (mesoporous)    -   Class III—pores in the order of 1-2 um.        The large pores Class III (micron order) were not obvious on        formulation #4012-2, although Class II pores did appear to be        present at this magnification.

The higher magnification images (45,000×) of these permeation layersdemonstrated that the fine pore structures of the mesoporous syntheticpolymer hydrogel permeation layers are very similar, with 4012-2,4012-3, and 4006-1 showing a mesoporous structure. The higher templateconcentration synthetic polymer hydrogel permeation layers 4012-3 and4006-1 also show micropores, separating areas of mesoporous permeationlayer material. Such micropores probably indicate the aggregation ofsurfactant micelles above a certain concentration in the polymerizationmixture, which would account for the sharp difference in morphologybetween 4012-2 and 4012-3, but relatively unchanged morphology between4012-3 and 4006-1.

Thus, the synthetic polymer hydrogel permeation layers may be classifiedin three groups: 4012-1 is nanoporous (Class I), 4012-2 is mesoporous(Class II), while 4012-3 and 4006-1 are mesoporous and microporous(Class II and III).

Example 3 Use of θ to Quantify the Porosity of Mesoporous SyntheticPolymer Hydrogel Permeation Layers

As is shown by the scanning electron micrographs, the porosity ofsynthetic polymer hydrogels can be varied by varying the amount oftemplate porogen, such as Brij surfactant, used in the polymerizationmixture. Although the SEM micrographs are useful to qualitatively andquantitatively evaluate the morphology of the mesoporous syntheticpolymer hydrogel permeation layers, a less labor intensive quantitativeporosity measurement was desired. Towards that end, dark fieldmicroscopy was used to quantify the morphological changes due to phaseseparation.

A dimensionless parameter, θ (theta), was used to express the degree ofphase separation, or porosity, based on light scattering measurementsunder the dark field microscope. The dimensionless degree of phaseseparation (θ) was determined by integrating the dark filed lightintensity readings a dry hydrogel layer on the test chip (λ), a standardlayer (λ_(S)) with a medium degree of phase separation and a non-phaseseparated, or solid, layer (λ₀) on the Leica INM 100 dark fieldmicroscope, and was computed with the following formula. When λ₀<<1 theequation can be simplified:$\theta \equiv \frac{\lambda - \lambda_{0}}{\lambda_{s} - \lambda_{0}} \approx \frac{\lambda}{\lambda_{s}}$An example of a surface which would approach the an ideal non-phaseseparated layer would be a very smooth surface, such as vapor-depositedplatinum on an electronics grade silicon wafer. The change in θ ofpolymer layers as a function of porosity was measured for 4012-1,4012-2, 4012-3, and 4006-1. The standard layer was a polyacrylamidehydrogel of the following composition (composition S):

Acrylamide: Bisacrylamide 19:1 (mol/mol)

Total monomer content 20% by weight

Under the illumination conditions used in the examples, λ_(S) was60±1.5, which was used for these experiments. The θ value for 4012-1,polymerized without template porogens, was 1.35. Addition of Brij to theformulation increases the phase separation or porosity, and thusincrease the θ value. Formulation 4012-2 had a θ of 3.06, 4012-3 had a θof 3.27 and 4006-1 had a theta of 3.00. Thus, all of the exemplarymesoporous synthetic polymer hydrogel permeation layers had θ's in therange 3.0-3.5.

Several micromolded permeation layers made from different batches ofpolymerization mixtures were tested using the above technique. Thepermeation layer formulation was the same as 4006-1, except thatN-acryl-SA was utilized instead of acryloyl-PEG-SA. The results areshown in the chart of FIG. 10. The θ porosity measurement is veryconsistent over twenty different batches (mean θ of 3.63±0.07).Interestingly, the use of N-acryl-SA, increased the relative phaseseparation, or porosity, of the permeation layer when compared toacyloyl-PEG-SA. This demonstrates the impact that subtle changes in thepolymerization formula can have on porosity. These data further showthat mesoporous synthetic hydrogel permeation layers can be molded ontoactive electronic matrix devices in a consistent fashion. Likewise, theconsistency of the light-scattering measurement technique is alsovalidated.

Example 4 Comparison of a Mesoporous Synthetic Polymer HydrogelPermeation Layer to a SA-Agarose Permeation Layer

In order to compare the performance of a mesoporous synthetic polymerhydrogel permeation layer to Nanogen's current SA-agarose permeationlayer (MSP), a single nucleotide polymorphism assay (SNP assay) wasperformed using active electronic matrix chips with the two layers. Inthe SNP1 assay, an ˜120 nt biotinylated amplicon, 5 nM in 50 mMhistidine, was electronically addressed to microlocations on both chipsat a constant potential of 2.1 V for 2 minutes. Cy 3 labeled reporteroligonucleotides for the C-allele and Cy 5 labeled reporteroligonucleotides for the T-allele, both 12 nt long, were then passivelyhybridized on the chip, at a concentration of 500 nM in a high saltbuffer (50 mM sodium phosphate, 500 mM sodium chloride).

The results are shown in FIG. 11. The mean fluorescent intensityreadings for a SNP 1 assay performed on three active electronic matrixchip device with 4006-1 permeation layers were generally above 2000 forall three chips tested. Both allele probes (C-allele—Cy-3, andT-allele—Cy-5) were detected well, allowing the easy identification ofheterozygous amplicon samples. As compared to the results of the sameassay performed on a standard SA-agarose permeation layer chip, the4006-1 mesoporous synthetic polymer hydrogel permeation layerformulation exhibited improved signal intensity in the SNP assay. Thefluorescence intensity readings on the 4006-1 chips were generallyhigher than those obtained using the SA-agarose chip.

Similar SNP assays of clinical interest were run on twenty 4006-1N-acryl-SA chips, using amplicons from sample nucleic acids of about 150to about 250 nt in length, and base-stacking format reporter probes. Ingeneral, biotinylated amplicons at relatively low concentrations wereelectronically addressed to microlocations on the active electronicmatrix chips in 50 mM histidine buffer. Stabilizer probes andbase-stacking stabilizable allele specific reporter probes (Cy-3 andCy-5 labeled) were then passively hybridized to the addressed ampliconsunder higher concentration and high-salt conditions. Mean fluorescentintensity measurements were then taken after thermal stringency wasapplied. The performance results for the assays are shown in the tablebelow:

Discrim- # of Correct Incorrect No ination SNP Genotype Exp. calls callscalls ratio SNP2 Mut/mut (red) 20 18 2 ≧21 Mut/wt 20 18 2 Wt/wt (green)20 18 2 ≧50 SNP3 Wt/wt (green) 20 18 2 ≧50 Wt/wt (green) 20 18 2 ≧40Mut/wt 20 18 2 SNP4 Wt/wt (green) 20 18 ≧100  Mut/mut (red) 20 20 ≧50Mut/wt 20 20 SNP5a Wt/wt (green) 14 14 ≧30 Wt/wt (green) 14 14 ≧30Mut/wt 14 14 Mut/wt 14 14 Wt/wt (green) 14 14 ≧30 Wt/wt (green) 14 14≧30 Mut/wt 14 14 SNP5b Wt/wt (green) 14 14 ≧30 Wt/wt (green) 14 14  ≧30vMut/wt 14 14 Mut/wt 14 14 Wt/wt (green) 14 14 ≧30 Wt/wt (green) 14 14≧30 Mut/wt 14 14 SNP6a Genotype A 20 20 Genotype B 20 20 ≧100  GenotypeC 20 20 ≧30 Genotype D 20 20 ≧60 SNP6b Genotype A 20 20 ≧70 Genotype B20 20 ≧60 Genotype C 20 20 ≧40

As can be seen from the results of the above table, the 4006-1 activeelectronic matrix chips performed very well in the SNP assays tested,with excellent discrimination ratios between alleles (Cy-3 and Cy-5labeled reporter oligos). SNP2 and SNP3 were tested in a multiplexedassay format, in which both amplicons were individually amplified from anucleic acid sample, and then simultaneously addressed to the samemicrolocation and tested by hybridization with the reporter oligomerssequentially. The fluorescence results were read sequentially, afterapplying thermal stringency to remove one set of reporter oligomers. Thetwo no-calls for SNP 2 were later verified to be amplification failurein the particular nucleic acid sample.

Example 5 Comparison of Small and Medium Length Nucleic Acid Binding inMesoporous Synthetic Polymer Hydrogel Permeation Layers

The commercially available Nanogen NanoChip® Molecular BiologyWorkstation (Nanogen, Inc.) chip addressing module is able to addressfour chips at the same time, under essentially the same conditions.Thus, for synthetic polymer hydrogel permeation layer comparisonexperiments, four chips with four different acryloyl-PEG-SA hydrogelformulations (4012-1, 4012-2, 4012-3, and 4006-1) were placed into theaddressing module and addressed side-by side according to the followingprocedures:

The ability of relatively small nucleic acids (under 50 nt in length) tobind to copolymerized SA attachment moieties in the mesoporous syntheticpolymer hydrogel permeation layers as compared to nanoporous syntheticpolymer hydrogel permeation layers (such as normal crosslinkedacrylamide hydrogels) was ascertained by a direct binding assay. This isa direct measure of the degree of the binding of relatively smallnucleic acids, as would be used for capture probes, onto the syntheticpolymer hydrogel permeation layer chips. A fluorophore labeled 46-ntDNA, biotinylated at the 5′ end, 5 nM, was addressed electronically tothe chip (2.0 v; 60 seconds). As the nucleic acids were directly labeledwith the fluorophore, fluorescence intensity on the chip was a directmeasure of the degree of DNA binding. The results of the assay are shownin the diamond-marked line in FIG. 12.

In order to measure the ability of medium sized nucleic acids (about60-150 nucleotides in length) to copolymerized SA attachment moieties inthe mesoporous synthetic polymer hydrogel permeation layers as comparedto nanoporous synthetic polymer hydrogel permeation layers, a dot-blotassay was used. A 114 nt single stranded DNA amplicon, biotinylated atthe 5′ end, 5 nM was electronically addressed to the chips under thesame electronic conditions. A fluorophore labeled reporteroligonucleotide (12 bp) was then hybridized passively to the ampliconsbound to the chip. The fluorescence intensity results for the differentsynthetic polymer hydrogel permeation layer formulations are shown bythe square-marked in FIG. 12.

Thus, nanoporous hydrogels such as #4012-1, seem to favor bindinginteractions with shorter oligonucleotides. The MFI readings for the114-mer were almost 3 fold less than that of a 46-mer. Binding of thelonger DNA (114-mer) increased drastically (˜4 fold) when a mesoporouspermeation layer was utilized (4012-2, 4012-3, and 4006-1). Increasedsignal intensity more directly correlates with increased porosity (θ)until a θ greater than about 3.0 (i.e., composition 4006-1). These datasuggest that maximal performance for this type of assay may be obtainedusing a mesoporous synthetic hydrogel permeation layer with a porositywith a θ from about 2 to about 3. In these data, the mesoporoussynthetic polymer hydrogel permeation layers showed a clear advantageover the nanoporous synthetic polymer hydrogel permeation layer whenaddressing amplicons of a size often used for SNP, STR, or geneexpression analysis. This may be the result of increased accessibilityof the immobilized SA to the long DNA via the larger pores.

The shorter DNA (46-bp), however, showed a slight decrease in bindingwith increased porosity. Results of additional experiments suggest that,under electronic addressing conditions, shorter DNA may permeate throughthe mesoporous synthetic polymer hydrogel permeation layer withouthaving sufficient time to achieve streptavidin-biotin complexation. Thishas further been confirmed by passive DNA binding experiments (i.e., theabsence of any applied electric field) wherein the short DNA boundequally well to both mesoporous and nonporous layers.

Example 6 Comparison of Primer Extension Reporting Linearity inMesoporous Synthetic Polymer Hydrogel Permeation Layers

In multiplexed assays in which the presence and/or quantity of a largenumber of amplicons are to be detected, primer extension detection ofthe amplicons is desirable to avoid reporter/stabilizer probedimerization. Thus, to test the four synthetic polymer hydrogelformulations, electronic hybridization of a target deoxyribonucleic acid(78 nt) was performed, followed by enzymatic reporting on the chips.Initially, a capture oligonucleotide (500 nM) complementary to thetarget DNA sequence was loaded on the chip, and subsequentlyelectronically hybridized to different concentrations (0.5 nM, 1.0 nM,2.0 nM, 4.0 nM, 8.0 nM, and 16.0 nM) of the target. Enzymatic primerextension using dNTPs containing dCTP-Cy5 was used to quantify thedegree of target hybridization.

In these assays, the fluorescence intensity measured on the chip isinfluenced by two main factors: the efficiency of binding of the DNA onthe chip, and the efficiency of capture primer extension by thepolymerase enzyme. The extension reaction depends on the porosity of thepolymer surface and subsequent accessibility to the DNA in the matrix.The results are shown in FIG. 13.

As shown in the FIGS. 13, the nanoporous synthetic polymer hydrogelpermeation layer, 4012-1, showed a relatively linear increase in MFI fortarget concentrations of up to ˜2 nM. Further increase in concentrationdid not increase the signal intensity. This phenomenon may be due to acombined effect of 1) a decrease in target hybridization and/or 2) adecrease in access to the hybridized nucleic acid by the polymeraseenzyme. In contrast, the mesoporous synthetic polymer hydrogelpermeation layers (4012-2, 4012-3, and 4006-1) exhibited a good linearincrease in signal intensity with increase in target concentration overthe entire concentration range spanning two orders of magnitude. Thistype of linear response curve is desirable for applications in whichquantitative relationships between the target nucleic acids are beingstudied, such as in gene expression analysis, or in viralload/population studies.

Example 7 Comparison of Various Synthetic Hydrogel Permeation LayerFormulations Using the Light-Scattering θ Technique and Reverse Dot-BlotNucleic Acid Assays

In order to explore the effect of various amounts of template porogen onthe porosity of the synthetic polymer hydrogel permeation layers, asmeasured by θ, permeation layers were formed on active electronic matrixchip substrates as described above using the basic polymerizationmixture with various amounts of Brij 700 surfactant added.Concentrations (mg/ml) used were: 0.0, 3.9, 9.3, 18.6, 55.8, 71.3, 80.6,102.3, and 170.4. As the critical micelle concentration for most Brijsurfactants is 1 mg/ml, 3.9 was the lowest concentration used. Thestandard λ used for the θ measurement was 60±1.5. As seen in FIG. 14,the synthetic polymer hydrogel permeation layers showed a rather markedincrease in phase separation, or porosity, throughout the lowerconcentrations of surfactant template. This leveled off at about 80mg/ml Brij 700 surfactant to a maximum θ range of about 3.5. This isconsistent with the observations of porosity by SEM examination of thepermeation layers, as described in Example 2.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it may be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A mesoporous synthetic polymer hydrogel permeation layer overlying anelectrode on a substrate, wherein the permeation layer comprisesmesopores which are between about 100 nm and 1000 nm across, wherein thepermeation layer is covalently anchored to the electrode and wherein theelectrode comprises a silicon-containing conductive material.
 2. Themesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the mesopores are between about 100 nm and about 500 nm across.3. The mesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the mesopores are between about 200 nm and about 500 nm across.4. The mesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the permeation layer further comprises micropores which arebetween about 1.0 μm and 3.0 μm across.
 5. The mesoporous syntheticpolymer hydrogel permeation layer of claim 4 wherein the micropores arebetween about 1.0 μm and 2.0 μm across.
 6. The mesoporous syntheticpolymer hydrogel permeation layer of claim 4 wherein the micropores arebetween about 1.0 μm and 1.5 μm across.
 7. The mesoporous syntheticpolymer hydrogel permeation layer of claim 1 wherein the permeationlayer is between about 0.5 μm and about 10 μm thick when dry.
 8. Themesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the permeation layer is between about 1.0 μm and about 5.0 μmthick when dry.
 9. The mesoporous synthetic polymer hydrogel permeationlayer of claim 1 wherein the permeation layer is between about 1.0 μmand about 2.0 μm thick when dry.
 10. The mesoporous synthetic polymerhydrogel permeation layer of claim 1 wherein the polymer comprises apolymerized acryloyl or acrylamido monomer.
 11. The mesoporous syntheticpolymer hydrogel permeation layer of claim 10 wherein the acryloyl oracrylamido monomer is an acrylamide.
 12. The mesoporous syntheticpolymer hydrogel permeation layer of claim 10 wherein the acryloyl oracrylamido monomer is an N-substituted acrylamide.
 13. The mesoporoussynthetic polymer hydrogel permeation layer of claim 10 wherein theacryloyl or acrylamido monomer is an N-substituted methacrylamide. 14.The mesoporous synthetic polymer hydrogel permeation layer of claim 10wherein the acryloyl or acrylamido monomer is methacrylamide.
 15. Themesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the permeation layer is covalently anchored to the electrode.16. The mesoporous synthetic polymer hydrogel permeation layer of claim1 wherein the electrode comprises a material selected from the groupconsisting of: platinum silicide, titanium silicide, gold silicide, andtungsten silicide.
 17. The mesoporous synthetic polymer hydrogelpermeation layer of claim 1 wherein the permeation layer is covalentlyanchored to the substrate around the electrode.
 18. The mesoporoussynthetic polymer hydrogel permeation layer of claim 17 wherein thepermeation layer is further covalently anchored to the electrode. 19.The mesoporous synthetic polymer hydrogel permeation layer of claim 1wherein the permeation layer comprises a copolymerized attachment moietyfor the attachment of specific binding entities.
 20. The mesoporoussynthetic polymer hydrogel permeation layer of claim 19 wherein thespecific binding entity is copolymerized with the synthetic polymerhydrogel through a structure of the general formula:

wherein, P is a polymerizable moiety covalently attached to one or twomoieties selected from the group consisting of: a monomeric unit of thesynthetic polymer and another P-X-R group, as defined herein, whereinthe other P-X-R group may be the same as or different from the firstP-X-R group, further wherein the dashed line is a covalent bond to thesecond moiety if P is covalently attached to two moieties; X is acovalent bond or a linking moiety; and R is a functional moiety forattaching, either covalently or non-covalently, a biomolecule.
 21. Themesoporous synthetic polymer hydrogel permeation layer of claim 20wherein R is attached to a biomolecule.
 22. The mesoporous syntheticpolymer hydrogel permeation layer of claim 21 wherein the biomolecule isa nucleic acid.
 23. The mesoporous synthetic polymer hydrogel permeationlayer of claim 21 wherein the biomolecule is a protein.
 24. Themesoporous synthetic polymer hydrogel permeation layer of claim 20wherein R is selected from the group consisting of biotin, avidin,streptavidin, and another biotin binding moiety.
 25. The mesoporoussynthetic polymer hydrogel permeation layer of claim 20 wherein R isstreptavidin.
 26. The mesoporous synthetic polymer hydrogel permeationlayer of claim 20 wherein R is selected from the group consisting ofaldehyde, carboxylic acid, acyl halide, succinimidyl, maleimidyl, thiol,hydrazide, hydrazine, amine, ester, thioester, ketal, and disulfidemoieties.
 27. The mesoporous synthetic polymer hydrogel permeation layerof claim 20 wherein P is selected from the group consisting ofacrylamide, acrylate, methacrylate, methacrylamide, allyl, amino, andepoxy moieties.
 28. The mesoporous synthetic polymer hydrogel permeationlayer of claim 20 wherein P is an acryloyl or acrylamido moiety.
 29. Themesoporous synthetic polymer hydrogel permeation layer of claim 20wherein X is a covalent bond.
 30. A mesoporous synthetic polymerhydrogel permeation layer overlying an electrode on a substrate, whereinthe permeation layer has a θ value between about 2.0 and about 4.0 whencompared to a standard permeation layer composition S, wherein saidcomposition comprises a ratio of Acrylamide: Bisacrylamide 19:1 mol/mol,total monomer content 20% by weight, wherein θ is defined by theequation:$\theta \equiv \frac{\lambda - \lambda_{0}}{\lambda_{s} - \lambda_{0}} \approx \frac{\lambda}{\lambda_{s}}$wherein integrated light intensity readings λ are taken using dark fieldmicroscopy on a compound optical microscope of the dry hydrogelpermeation layer on the substrate (λ), the standard permeation layer(λ_(s)) and a non-phase separated or solid layer (λ₀).
 31. Themesoporous synthetic polymer hydrogel permeation layer of claim 30wherein the permeation layer has a θ value between about 2.0 and about3.8 when compared to composition S.
 32. The mesoporous synthetic polymerhydrogel permeation layer of claim 30 wherein the permeation layer has aθ value between about 2.0 and about 3.0 when compared to composition S.33. The mesoporous synthetic polymer hydrogel permeation layer of claim30 wherein the permeation layer has a θ value between about 3.0 andabout 3.8 when compared to composition S.
 34. The mesoporous syntheticpolymer hydrogel permeation layer of claim 30 wherein the permeationlayer is between about 0.5 μm and about 10 μm thick when dry.
 35. Themesoporous synthetic polymer hydrogel permeation layer of claim 30wherein the permeation layer is between about 1.0 μm and about 5.0 μmthick when dry.
 36. The mesoporous synthetic polymer hydrogel permeationlayer of claim 30 wherein the permeation layer is between about 1.0 μmand about 2.0 μm thick when dry.
 37. The mesoporous synthetic polymerhydrogel permeation layer of claim 30 wherein the polymer comprises apolymerized acryloyl or acrylamido monomer.
 38. The mesoporous syntheticpolymer hydrogel permeation layer of claim 37 wherein the acryloyl oracrylamido monomer is an acrylamide.
 39. The mesoporous syntheticpolymer hydrogel permeation layer of claim 37 wherein the acryloyl oracrylamido monomer is an N-substituted acrylamide.
 40. The mesoporoussynthetic polymer hydrogel permeation layer of claim 37 wherein theacryloyl or acrylamido monomer is an N-substituted methacrylamide. 41.The mesoporous synthetic polymer hydrogel permeation layer of claim 37wherein the acryloyl or acrylamido monomer is methacrylamide.
 42. Themesoporous synthetic polymer hydrogel permeation layer of claim 30wherein the permeation layer is covalently anchored to the electrode.43. The mesoporous synthetic polymer hydrogel permeation layer of claim42 wherein the electrode comprises a silicon-containing conductivematerial.
 44. The mesoporous synthetic polymer hydrogel permeation layerof claim 43 wherein the electrode comprises a material selected from thegroup consisting of: platinum silicide, titanium silicide, goldsilicide, and tungsten silicide.
 45. The mesoporous synthetic polymerhydrogel permeation layer of claim 30 wherein the permeation layer iscovalently anchored to the substrate around the electrode.
 46. Themesoporous synthetic polymer hydrogel permeation layer of claim 45wherein the permeation layer is further covalently anchored to theelectrode.
 47. The mesoporous synthetic polymer hydrogel permeationlayer of claim 30 wherein the permeation layer comprises a copolymerizedattachment moiety for the attachment of specific binding entities. 48.The mesoporous synthetic polymer hydrogel permeation layer of claim 47wherein the specific binding entity is copolymerized with the syntheticpolymer hydrogel through a structure of the general formula:

wherein, P is a polymerizable moiety covalently attached to one or twomoieties selected from the group consisting of: a monomeric unit of thesynthetic polymer and another P-X-R group, as defined herein, whereinthe other P-X-R group may be the same as or different from the firstP-X-R group, further wherein the dashed line is a covalent bond to thesecond moiety if P is covalently attached to two moieties; X is acovalent bond or a linking moiety; and R is a functional moiety forattaching, either covalently or non-covalently, a biomolecule.
 49. Themesoporous synthetic polymer hydrogel permeation layer of claim 48wherein R is attached to a biomolecule.
 50. The mesoporous syntheticpolymer hydrogel permeation layer of claim 49 wherein the biomolecule isa nucleic acid.
 51. The mesoporous synthetic polymer hydrogel permeationlayer of claim 49 wherein the biomolecule is a protein.
 52. Themesoporous synthetic polymer hydrogel permeation layer of claim 48wherein R is selected from the group consisting of biotin, avidin,streptavidin, and another biotin binding moiety.
 53. The mesoporoussynthetic polymer hydrogel permeation layer of claim 48 wherein R isstreptavidin.
 54. The mesoporous synthetic polymer hydrogel permeationlayer of claim 48 wherein R is selected from the group consisting ofaldehyde, carboxylic acid, acyl halide, succinimidyl, maleimidyl, thiol,hydrazide, hydrazine, amine, ester, thioester, ketal, and disulfidemoieties.
 55. The mesoporous synthetic polymer hydrogel permeation layerof claim 48 wherein R is a hydrazide moiety.
 56. The mesoporoussynthetic polymer hydrogel permeation layer of claim 48 wherein R is asuccinimidyl ester moiety.
 57. The mesoporous synthetic polymer hydrogelpermeation layer of claim 48 wherein R is an aldehyde moiety.
 58. Themesoporous synthetic polymer hydrogel permeation layer of claim 48wherein R is a psoralen moiety.
 59. The mesoporous synthetic polymerhydrogel permeation layer of claim 48 wherein P is selected from thegroup consisting of acrylamide, acrylate, methacrylate, methacrylamide,allyl, amino, and epoxy moieities.
 60. The mesoporous synthetic polymerhydrogel permeation layer of claim 48 wherein P is selected an acryloylor acrylamido moiety.
 61. The mesoporous synthetic polymer hydrogelpermeation layer of claim 48 wherein X is a covalent bond.
 62. Themesoporous synthetic polymer hydrogel permeation layer of claim 48wherein X is a polyalkylene glycol linker.
 63. A mesoporous syntheticpolymer hydrogel permeation layer overlying an electrode on a substrate,wherein the permeation layer comprises mesopores which are between about100 nm and 1000 nm across, wherein the permeation layer comprises acopolymerized attachment moiety for the attachment of specific bindingentities, and wherein the specific binding entities are copolymerizedwith the permeation layer according to a structure of the

general formula: wherein, P is a polymerizable moiety covalentlyattached to one or two moieties selected from the group consisting of: amonomeric unit of the synthetic polymer and another P-X-R group, asdefined herein, wherein the other P-X-R group may be the same as ordifferent from the first P-X-R group, further wherein the dashed line isa covalent bond to the second moiety if P is covalently attached to twomoieties; X is a covalent bond or a linking moiety; and R is afunctional moiety for attaching, either covalently or non-covalently, abiomolecules, wherein R is selected from the group consisting of ahydrazide, succinimidyl ester, aldehyde, and psoralen moiety.
 64. Themesoporous synthetic polymer hydrogel permeation layer of claim 63wherein P is selected from the group consisting of acrylamide, acrylate,methacrylate, methacrylamide, allyl, amino, and epoxy moieties.
 65. Themesoporous synthetic polymer hydrogel permeation layer of claim 63wherein P is an acryloyl or acrylamido moiety.
 66. The mesoporoussynthetic polymer hydrogel permeation layer of claim 63 wherein X is acovalent bond.
 67. The mesoporous synthetic polymer hydrogel permeationlayer of claim 63 wherein R is attached to a biomolecule.
 68. Themesoporous synthetic polymer hydrogel permeation layer of claim 67wherein the biomolecule is a nucleic acid.
 69. The mesoporous syntheticpolymer hydrogel permeation layer of claim 67 wherein the biomolecule isa protein.
 70. A mesoporous synthetic polymer hydrogel permeation layeroverlying an electrode on a substrate, wherein the permeation layercomprises mesopores which are between about 100 nm and 1000 nm across,wherein the permeation layer comprises a copolymerized attachment moietyfor the attachment of specific binding entities, and wherein thespecific binding entities are copolymerized with the permeation layeraccording to a structure of the general formula:

wherein, P is a polymerizable moiety covalently attached to one or twomoieties selected from the group consisting of: a monomeric unit of thesynthetic polymer and another P-X-R group, as defined herein, whereinthe other P-X-R group may be the same as or different from the firstP-X-R group, further wherein the dashed line is a covalent bond to thesecond moiety if P is covalently attached to two moieties; X is apolyalkylene glycol linker; and R is a functional moiety for attaching,either covalently or non-covalently, a biomolecules.
 71. The mesoporoussynthetic polymer hydrogel permeation layer of claim 70 wherein P isselected from the group consisting of acrylamide, acrylate,methacrylate, methacrylamide, allyl, amino, and epoxy moieties.
 72. Themesoporous synthetic polymer hydrogel permeation layer of claim 70wherein P is an acryloyl or acrylamido moiety.
 73. The mesoporoussynthetic polymer hydrogel permeation layer of claim 70 wherein X is acovalent bond.
 74. The mesoporous synthetic polymer hydrogel permeationlayer of claim 70 wherein R is attached to a biomolecule.
 75. Themesoporous synthetic polymer hydrogel permeation layer of claim 74wherein the biomolecule is a nucleic acid.
 76. The mesoporous syntheticpolymer hydrogel permeation layer of claim 74 wherein the biomolecule isa protein.
 77. The mesoporous synthetic polymer hydrogel permeationlayer of claim 70 wherein R is selected from the group consisting ofbiotin, avidin, streptavidin, and another biotin binding moiety.
 78. Themesoporous synthetic polymer hydrogel permeation layer of claim 70wherein R is streptavidin.
 79. The mesoporous synthetic polymer hydrogelpermeation layer of claim 70 wherein R is selected from the groupconsisting of aldehyde, carboxylic acid, acyl halide, succinimidyl,maleimidyl, thiol, hydrazide, hydrazine, amine, ester, thioester, ketal,and disulfide moieties.
 80. The mesoporous synthetic polymer hydrogelpermeation layer of claim 70 wherein R is selected from the groupconsisting of a hydrazide, succinimidyl ester, aldehyde, and psoralenmoiety.